Mycobacterial antigens expressed during latency

ABSTRACT

A method is provided for identifying mycobacterial genes that are induced or up-regulated under culture conditions that are nutrient-starving and which maintain mycobacterial latency, said conditions being obtainable by batch fermentation of a  micobacterium  for at least 20 days post-inoculation, when compared with culture conditions that are not nutrient-starving and which support exponential growth of said  mycobacterium . Said induced or up-regulated genes form the basis of nucleic acid vaccines, or provide targets to allow preparation of attenuated  mycobacteria  for vaccines against mycobacterial infections. Similarly, peptides encoded by said induced or up-regulated genes are employed in vaccines. In a further embodiment, the identified genes/peptides provide the means for identifying the presence of a mycobacterial infection in a clinical sample by nucleic acid probe or antibody detection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Phase of International PublicationNo. WO 03/004520 A2, filed as PCT/GB02/03052 on Jul. 4, 2002, whichclaims priority to GB 0116385.6, filed Jul. 4, 2001, and GB 0123993.8,filed Oct. 5, 2001.

The present invention relates to a method of identifying a gene inmycobacteria the expression of which is induced or up-regulated duringmycobacterial latency, to the isolated peptide products, variants,derivatives or fragments thereof, to antibodies, that bind to saidpeptides, variants, derivatives or fragments, to DNA and RNA vectorsthat express said peptides variants, derivatives or fragments, toattenuated mycobacteria in which the activity of at least one of saidinduced or up-regulated genes has been modified, to vaccines againstmycobacterial infections, and to methods of detecting the presence of amycobacterial infection.

Many microorganisms are capable of forming intracellular infections.These include: infections caused by species of Salmonella, Yersinia,Shigella, Campylobacter, Chlamydia and Mycobacteria. Some of theseinfections are exclusively intracellular, others contain bothintracellular and extracellular components. However, it is theintracellular survival cycle of bacterial infection which is suspectedas a main supportive factor for disease progression.

Generally, these microorganisms do not circulate freely in the body, forexample, in the bloodstream, and are often not amenable to drugtreatment regimes. Where drugs are available, this problem has beenexacerbated by the development of multiple drug resistantmicroorganisms.

A number of factors have contributed to the problem of microbialresistance. One is the accumulation of mutations over time and thesubsequent horizontal and vertical transfer of the mutated genes toother organisms. Thus, for a given pathogen, entire classes ofantibiotics have been rendered inactive. A further factor has been theabsence of a new class of antibiotics in recent years. The emergence ofmultiple drug-resistant pathogenic bacteria represents a serious threatto public-health and new forms of therapy are urgently required.

For similar reasons, vaccine therapies have not proved effective againstsuch intracellular microorganisms. Also, increased systemicconcentration of antibiotics to improve bioavailability within cells mayresult in severe side effects.

Mycobacterium tuberculosis (TB) and closely related species make up asmall group of mycobacteria known as the Mycobacterium tuberculosiscomplex (MTC). This group comprises four species M. tuberculosis, M.microti, M. bovis and M. africanum which are the causative agent in themajority of tuberculosis (TB) cases throughout the world.

M. tuberculosis is responsible for more than three million deaths a yearworld-wide. Other mycobacteria are also pathogenic in man and animals,for example M. avium subsp. paratuberculosis which causes Johne'sdisease in ruminants, M. bovis which causes tuberculosis in cattle, M.avium and M. intracellulare which cause tuberculosis inimmunocompromised patients (eg. AIDS patients, and bone marrowtransplant patients) and M. leprae which causes leprosy in humans.Another important mycobacterial species is M. vaccae.

M. tuberculosis infects macrophage cells within the body. Soon aftermacrophage infection, most M. tuberculosis bacteria enter and replicatewithin cellular phagosome vesicles, where the bacteria are sequesteredfrom host defences and extracellular factors.

It is the intracellular survival and multiplication or replication ofbacterial infection which is suspected as a main supportive factor formycobacterial disease progression.

A number of drug therapy regimens have been proposed for combatting M.tuberculosis infections, and currently combination therapy including thedrug isoniazid has proved most effective. However, one problem with suchtreatment regimes is that they are long-term, and failure to completesuch treatment can promote the development of multiple drug resistantmicroorganisms.

A further problem is that of providing an adequate bioavailability ofthe drug within the cells to be treated. Whilst it is possible toincrease the systemic concentration of a drug (eg. by administering ahigher dosage) this may result in severe side effects caused by theincreased drug concentration.

The effectiveness of vaccine prevention against M. tuberculosis hasvaried widely. The current M. tuberculosis vaccine, BCG, is anattenuated strain of M. bovis. It is effective against severecomplications of TB in children, but it varies greatly in itseffectiveness in adults particularly across ethnic groups, BCGvaccination has, been used to prevent tuberculous meningitis and helpsprevent the spread of M. tuberculosis to extra-pulmonary sites, but doesnot prevent infection.

The limited efficacy of BCG and the global prevalence of TB has led toan international effort to generate new, more effective vaccines. Thecurrent paradigm is that protection will be mediated by the stimulationof a Th1 immune response.

BCG vaccination in man was given orally when originally introduced, butthat route was discontinued because of loss of viable BCG during gastricpassage and of frequent cervical adenopathy. In experimental animalspecies, aerosol or intra-tracheal delivery of BCG has been achievedwithout adverse effects, but has varied in efficacy from superiorprotection than parenteral inoculation in primates, mice; and guineapigs to no apparent advantage over the subcutaneous route in otherstudies.

There is therefore a need for an improved and/or alternative vaccine ortherapeutic agent for combatting mycobacterial infections.

An additional major problem associated with the control of mycobacterialinfections, especially M. tuberculosis infections, is the presence of alarge reservoir of asymptomatic individuals infected with mycobacteria.Dormant mycobacteria are even more resistant to front-line drugs.

Infection with mycobacteria (eg. M. tuberculosis) rarely leads to activedisease, and most individuals develop a latent infection which maypersist for many years before reactivating to cause disease (Wayne,1994). The current strategy for controlling such infection is earlydetection and treatment of patients with active disease. Whilst this isessential to avoid deaths and control transmission, it has no effect oneliminating the existing reservoir of infection or on preventing newcases of disease through reactivation.

Conventional mycobacterial vaccines, including BCG, protect againstdisease and not against infection. Ideally a new mycobacterial vaccinewill impart sterile immunity, and a post-exposure vaccine capable ofboosting the immune system to kill latent mycobacteria or preventreactivation to active disease-causing microorganisms would also bevaluable against latent infection.

Conventional detection of latent mycobacterial infection by skin testingmay be compromised. For example, current TB detection methods based ontuberculin skin testing are compromised by BCG vaccination and byexposure to environmental mycobacteria.

New strategies are therefore required for more effective diagnosis,treatment and prevention of mycobacterial latent infection.

To develop specific strategies for addressing latent mycobacterialinfection it is necessary to elucidate the physiological, biochemicaland molecular properties of these microorganisms.

At present, there is no suitable in vivo model for studyingmycobacterial latent infection and such a model is unlikely to providesufficient microbial material to enable detailed analysis of thephysiological and molecular changes that occur.

Studies to date have used either static cultures which allow tuberclebacilli to generate oxygen-depletion gradients and enter anon-replicating persistent state in the sediment layer, or agitatedsealed liquid cultures (Wayne and Lin, 1982; Cunningham and Spreadbury,1998; Wayne and Hayes, 1996). Transition to a non-replicating persistentstate in these models coincides with a shift-down to glyoxylatemetabolism, resistance to isoniazid and rifampicin and susceptibility tothe anaerobic bactericidal action of metronidazole (Wayne and Hayes,1996).

For example, a number of publications have described the analysis ofmycobacterial gene and protein expression profiles following exposure ofthe mycobacteria to various environmental stimuli. These includeSherman, D. R. et al (2001) PNAS, vol. 98, no. 13, pp.7534-7539; Hutter,B. (2000) FEMS Microbiol. Letts. 188, pp. 141-146; Michele, T. M. et al.(1999) Antimicrobial Agents and Chemotherapy, vol. 43, no. 2, pp.216-225; Yuan, Y. et al. (1998) PNAS, vol. 95, pp. 9578-9583; Boon, C.et al (2001) J. Bacteriol., vol. 183, no. 8, pp. 2672-2676; Cunningham,A. F. et al (1998) J. Bacteriol., vol. 180, no. 4, pp. 801-808;Murugasu-Oei, B. et al (1999) Mol. Gen. Genet., vol. 262, pp. 677-682;and a number of patent publications such as WO99/24067, WO99/04005,WO97/35611, and WO92/08484. The mycobacteria employed in these analyseshave been grown in crude, batch systems, with the result that there islittle or no control of the environmental stimuli to which themycobacteria have been exposed. Accordingly, the bacteria experience alarge number of complex, interactive environmental stimuli, some ofwhich may have rapid and transient effects in terms of gene and proteinexpression.

Such studies are poorly defined and controlled; and experiments relyingon self-generated oxygen-depletion gradients have yielded inconsistentresults. In addition, the described studies have been conducted over arelatively short duration in terms of post-inoculation growth, in manycases up to approximately 2 weeks post-inoculation, with the result thatthe cultured bacteria are exposed to environmental stimuli associatedwith the mid to late exponential phase, and/or the early stationaryphase.

In view of the above, there is a need for a defined and controlled modelfor studying mycobacterial (eg. TB) persistence which simulates keyfeatures of the in vivo environment.

According to a first aspect of the present invention there is providedan isolated mycobacterial peptide, or a fragment or derivative orvariant of said peptide, wherein the peptide is encoded by amycobacterial gene the expression of which is induced or up-regulatedunder culture conditions that are nutrient-starving and which maintainmycobacterial latency, said conditions being obtainable by batchfermentation of a micobacterium for at least 20 days post-inoculation,when compared with culture conditions that are not nutrient-starving andwhich support exponential growth of said mycobacterium.

Latency is synonymous with persistence. These terms describe areversible state of low metabolic activity in which mycobacterial cellscan survive for extended periods without cell division.

In contrast to the various prior art analyses, the present invention isconcerned with the induction or up-regulation of mycobacterial genes(and the corresponding gene products) during long term latencyconditions rather than during the onset of latency (ie. late exponentialphase, or early stationary phase).

The preferred culture method of the present invention is that of batchfermenter culture. This method permits careful monitoring and control ofgrowth culture parameters such as pH, temperature, available nutrients,and dissolved oxygen tension (DOT). In particular, temperature and DOTmay be strictly controlled. In contrast, careful monitoring and controlis not possible with convention, crude batch culture systems, with theresult that mycobacteria cultured by such systems are exposed to amultiplicity of complex, interactive environmental stimuli, some ofwhich may have rapid and transient effects in terms of gene and proteinexpression. Thus, the batch fermenter system of the present inventionallows relatively careful control of environmental stimuli so that amycobacterial response to a particular stimulus (eg. nutrientstarvation) can be analyzed in relative isolation from otherenvironmental stimuli that may otherwise obscure or modify theparticular mycobacterial response of interest.

In use of the present method it is possible to ensure that the principallatency induction parameter employed is starvation of carbon, andpreferably the starvation of carbon and energy. This means that theaccidental induction or up-regulation of genes that are solelyresponsive to other environmental switches may be substantiallyprevented. Accordingly, false-positive identification of genes that areinduced or up-regulated under conditions unrelated to carbon; starvationand/or energy limitation may be substantially avoided.

The term “nutrient-starving” in the context of the present inventionmeans that the concentration of the primary carbon, and preferably theprimary energy source, is insufficient to support growth of themycobacteria. “Nutrient-starving” is a term associated with anestablished mid to late stationary phase of a batch culture growthcurve. Under such conditions the mycobacteria are metabolicallystressed, rather than simply reduced in growth rate.

In more detail, exponential growth is that period of growth which isassociated with a logarithmic increase in mycobacterial cell mass (alsoknown as the “log” phase) in which the bacteria are multiplying at amaximum specific growth rate for the prevailing culture conditions.During this period of growth the concentrations of essential nutrientsdiminish and those of end products increase. However, once the primarycarbon and/or primary energy source falls to below a critical level, itis no longer possible for all of the mycobacterial cells within theculture to obtain sufficient carbon and/or energy needed to supportoptimal cellular function and cell division. Once this occurs,exponential growth slows and the mycobacteria enter stationary phase.Thereafter, the mycobacteria become nutrient starved, and enter latency.It is this latent state in the growth phase, rather than the lateexponential phase or early stationary phase, with which the presentinvention is concerned.

Carbon starvation refers to a growth state in which the concentration ofexogenous carbon is insufficient to enable the bacteria to grow and orreplicate. However, when in this state, there may be other energysources. (eg. endogenous reserves, secondary metabolites) that areavailable to maintain essential cellular functions and viability withoutsupporting growth. Thus, carbon starvation is associated with a mid orlate stationary phase condition in which the exogenous carbon source hasbecome depleted and bacterial growth has substantially ceased. In termsof a batch fermenter culture of mycobacteria, this typically occurs at20 days (or later) post inoculation.

The onset of stationary phase vis-a-vis the time of inoculation willdepend on a number of factors such as the particular mycobacterialspecies/strain, the composition of the culture media (eg. the particularprimary carbon and energy source), and the physical culture parametersemployed.

However, as a guide, the end of exponential phase and the onset ofstationary phase generally corresponds to that point in the growth phaseassociated with the maximum number of viable counts of mycobacteria.

In use of the present invention, the exponential phase mycobacterialcells are harvested from the culture vessel at a point in the growthphase before the maximum number of total viable counts has beenachieved. This point in the growth phase may be mimicked undercontinuous culture-conditions employing a steady state growth rateapproximating μ_(max) and providing a generation time of approximately18-24 hours. In a preferred embodiment, the exponential phasemycobacterial cells are harvested when a value of between 2 and 0.5(more preferably between 1 and 0.5) log units of viable counts per ml ofculture medium less than the maximum number of viable counts per ml ofculture medium has been achieved. Thus, the “exponential” phase cellsare generally harvested during mid-log phase.

For example, if the maximum viable count value is 1×10¹⁰ per ml, thenthe “exponential” phase cells would be preferably harvested once a valueof between 1×10⁸ and 1×10^(9.5) (more preferably between 1×10⁹ and1×10^(9.5)) viable counts per ml has been achieved. In the case of M.tuberculosis, this would be approximately 3-10, preferably 4-7 dayspost-inoculation.

In use of the present invention, the nutrient-starved, batch fermentercultured mycobacterial cells are harvested from the culture vessel at apoint in the growth phase after the maximum number of total viablecounts has been achieved. This point in the growth phase may be mimickedunder continuous culture conditions supporting a generation time of atleast 3 days. In a preferred embodiment, the stationary phasemycobacterial cells are harvested when the viable counts per ml ofculture medium has fallen by at least 0.5, preferably at least 1, morepreferably at least 2 log units less than the maximum number of viablecounts per ml of culture medium. Thus, the nutrient-starved cells aregenerally harvested during mid- to late-stationary phase.

For example, if the maximum viable count value is 1×10¹⁰ per ml, thenthe stationary phase cells would be preferably harvested once the viablecount number had fallen to a value of at least 1×10^(9.5), preferably atleast 1×10⁹, more preferably at least 1×10⁸ viable counts per ml. In thecase of M. tuberculosis, this would be approximately at least day 20,preferably at least day 30, typically day 40-50 post-inoculation. Longerpost-inoculation harvesting times of at least 100 days, even at least150 days may be employed. For mycobacteria generally, the mid to latestationary phase cells are preferably harvested at least 20 days,preferably at least 30 days, more preferably at least 40 dayspost-inoculation.

Suitable media for culturing mycobacteria are described in Wayne, L. G.(1994) [in Tuberculosis: Pathogenesis, Protection, and Control publishedby the American Society for Microbiology, pp. 73-83]. These includeMiddlebrook 7H9 Medium [see Barker, L. P., et al. (1998) Molec.Microbiol., vol. 29(5), pp. 1167-1177], and WO00/52139 in the name ofthe present Applicant.

In use of the batch fermenter culture method, the starting concentrationof the primary carbon source (and preferably the primary energy source)is at least 0.5, preferably at least 1 gl⁻¹ of culture medium. Suchconcentrations are considered to be not nutrient-starving. Conversely,“nutrient-starving” conditions are associated with a primary carbon andenergy source concentration of less than 0.5, preferably less than 0.2,and more preferably less than 0.1 gl⁻¹ of culture medium. The preferredcarbon and energy source is glycerol.

In a preferred embodiment, the starting concentration of glycerol is atleast 1, preferably 1-3, more preferably approximately 2 gl⁻¹ of culturemedium. The onset of “nutrient-starving” conditions is associated with aconcentration of less than 0.2, preferably less than 0.1 gl⁻¹ of culturemedium.

Other primary carbon and energy sources may be employed such as glucose,pyruvate, and fatty acids (eg. palmitate, and butyrate). These sourcesmay be employed at substantially the same concentrations as forglycerol.

The pH of the culture medium is preferably maintained between pH 6 and8, more preferably between pH 6.5 and 7.5, most preferably at about pH6.9.

In one embodiment, the dissolved oxygen tension (DOT) is maintainedthroughout the culture process at at least 40% air saturation, morepreferably between 50 and 70% air saturation, most preferably at 50% airsaturation.

The dissolved oxygen tension parameter is calculated by means of anoxygen electrode and conventional laboratory techniques. Thus, 100% airsaturation corresponds to a solution that is saturated with air, whereas0% corresponds to a solution that has been thoroughly purged with aninert gas such as nitrogen. Calibration is performed under standardatmospheric pressure conditions and measured at 37° C., and withconventional air comprising approximately 21% oxygen.

In another embodiment of the present invention, latency may be inducedby a combination of carbon and/or energy source starvation, and a lowDOT.

In a preferred embodiment, the DOT is maintained at at least 40% airsaturation, more preferably between 50 and 70% air saturation, until themycobacterial culture has entered early-mid log phase. The DOT may bethen lowered so as to become limiting, for example in increments over a5 or 6 day period, and the culture maintained at a DOT of 0-10,preferably at a DOT of approximately 5% until the stationary phase cellsare harvested.

The carbon and energy starvation, and optional low oxygen tensionlatency induction conditions of the present invention are cultureconditions-that are conducive for a mycobacterium to express at leastone gene which would be normally expressed in vivo during latency of themycobacterium's natural target environment which is believed to involvea low carbon and energy, and low oxygen environment.

The mycobacterium is selected from the species M. phlei, M. smegmatis,M. africanum, M. caneti, M. fortuitum, M. marinum, M. ulcerans, M.tuberculosis, M. bovis, M. microti, M. avium, M. paratuberculosis, M.leprae, M. lepraemurium, M. intracellulare, M. scrofulaceum, M. xenopi,M. genavense, M. kansasii, M. simiae, M. szulgai, M. haemophilum, M.asiaticum, M. malmoense, M. vaccae and M. shimoidei. Of particularinterest are members of the MTC, preferably M. tuberculosis.

In use, it is preferred that those genes (ie. as represented by cDNAs inthe detection assay) which are up-regulated by at least 1.5-fold understationary phase conditions vis-a-vis exponential phase conditions areselected. In more preferred embodiments, the corresponding up-regulationselection criterium is at least 2-fold, more preferably 3-fold, mostpreferably 4-fold. In further embodiments up-regulation levels of atleast 10-fold, preferably 50-fold may be employed.

The term peptide throughout this specification is synonymous withprotein.

Use of mycobacterial peptide compositions, which peptides are associatedwith mycobacterial latency, provide excellent vaccine candidates fortargeting latent mycobacteria in asymptomatic patients infected withmycobacteria.

The terms “isolated,” “substantially pure,” and “substantiallyhomogenous” are used interchangeably to describe a peptide which hasbeen separated from components which naturally accompany it. A peptideis substantially pure when at least about 60 to 75% of a sample exhibitsa single peptide sequence. A substantially pure peptide will typicallycomprise about 60 to 90% w/w of a protein sample, more usually about95%, and preferably will be over about 99% pure. Peptide purity orhomogeneity may be indicated by, for example, polyacrylamide gelelectrophoresis of a protein sample, followed by visualizing a singlepolypeptide band upon staining the gel. Alternatively, higher resolutionmay be provided by using, for example, HPLC.

A peptide is considered to be isolated when it is separated from thecontaminants which accompany it in its natural state. Thus, a peptidewhich is chemically synthesized or synthesized in a cellular systemdifferent from the cell from which it naturally originates will besubstantially free from its naturally associated components.

The present invention provides peptides which may be purified frommycobacteria as well as from other types of cells transformed withrecombinant nucleic acids encoding these peptides.

If desirable, the amino acid sequence of the proteins of the presentinvention may be determined by protein sequencing methods.

The terms “peptide”, “oligopeptide”, “polypeptide”, and “protein” areused interchangeably and do not refer to a specific length of theproduct. These terms embrace post-translational modifications such asglycosylation, acetylation, and phosphorylation.

The term “fragment” means a peptide having at least five, preferably atleast ten, more preferably at least twenty, and most preferably at leastthirty-five amino acid residues of the peptide which is the gene productof the induced or up-regulated gene in question. The fragment preferablyincludes an epitope of the gene product in question.

The term “variant” means a peptide or peptide “fragment” having at leastseventy, preferably at least eighty, more preferably at least ninetypercent amino acid sequence homology with the peptide that is the geneproduct of the induced or up-regulated gene in question. An example of a“variant” is a peptide or peptide fragment of an induced/up-regulatedgene which contains one or more analogues of an amino acid (eg. anunnatural amino acid), or a substituted linkage. The terms “homology”and “identity” are considered synonymous in this specification. In afurther embodiment, a “variant” may be a mimic of the peptide or peptidefragment, which mimic reproduces at least one, epitope of the peptide orpeptide fragment. The mimic may be, for example, a nucleic acid mimic,preferably a DNA mimic.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences may be compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequent coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percentage sequence identityfor the test sequence(s) relative to the reference sequence, based onthe designated program parameters.

Optimal alignment of sequences for comparison may be conducted, forexample, by the local homology alignment algorithm of Smith and Waterman[Adv. Appl. Math. 2: 484 (1981)], by the algorithm of Needleman & Wunsch[J. Mol. Biol. 48: 443 (1970)] by the search for similarity method ofPearson & Lipman (Proc. Nat'l. Acad. Sci. USA 85: 2444 (1988)], bycomputer implementations of these algorithms (GAP, BESTFIT, FASTA, andTFASTA-Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 University.Avenue, Madison, Wis., 53705), or by visual inspection [see CurrentProtocols in Molecular Biology, F. M. Ausbel et al, eds, CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc. (1995 Supplement) Ausbubel].

Examples of algorithms suitable for determining percent sequencesimilarity are the BLAST and the BLAST 2.0 algorithms [see Altschul(1990) J. Mol. Biol. 215: pp. 403-410].

In a preferred homology comparison, the identity exists over a region ofthe sequences that is at least 10 amino acid residues in length.

The term “derivative” means a peptide comprising the peptide (orfragment, or variant thereof) which is the gene product of the inducedor up-regulated gene in question. Thus, a derivative may include thepeptide in question, and a further peptide sequence which may introduceone or more additional epitopes.

The further peptide sequence should preferably not interfere with thebasic folding and thus conformational structure of the peptide inquestion. Examples of a “derivative” are a fusion protein, a conjugate,and a graft. Thus, two or more peptides (or fragments, or variants) maybe joined together to form a derivative. Alternatively, a peptide (orfragment, or variant) may be joined to an unrelated molecule (eg. apeptide). Derivatives may be chemically synthesized, but will betypically prepared by recombinant nucleic acid methods. Additionalcomponents such as lipid, and/or polysaccharide, and/or polyketidecomponents may be included.

All of the molecules “fragment”, “variant” and “derivative” have acommon antigenic cross-reactivity and/or substantially the same in vivobiological activity as the gene product of the induced or up-regulatedgene in question from which they are derived. For example, an antibodycapable of binding to a fragment, variant or derivative would be alsocapable of binding to the gene product of the induced or up-regulatedgene in question. It is a preferred feature that the fragment, variantand derivative each possess the active site of the peptide which is theinduced or up-regulated peptide in question. Alternatively, all of theabove embodiments of a peptide of the present invention share a commonability to induce a “recall response” of a T-lymphocyte which has beenpreviously exposed to an antigenic component of a mycobacterialinfection.

In a preferred embodiment, the peptide is selected from the groupconsisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153,155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181,183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209,211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237,239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265,267, 269, 271, 273, 275, 277, 279 and 281.

According to a second aspect of the invention there is provided a methodof identifying a mycobacterial gene the expression of which is inducedor up-regulated during mycobacterial latency, said method comprising:—

-   -   culturing a first micobacterium under culture conditions that        are nutrient-starving and which maintain mycobacterial latency,        said conditions being obtainable by batch fermentation of the        first micobacterium for at least 20 days post-inoculation;    -   culturing a second micobacterium under culture conditions that        are not nutrient-starving and which support exponential growth        of the second mycobacterium;    -   obtaining first and second mRNA populations from said first and        second mycobacteria respectively, wherein said first mRNA        population is obtained from the first micobacterium which has        been cultured under nutrient-starving conditions obtainable by        batch fermentation of the first micobacterium for at least 20        days post-inoculation, and wherein said second mRNA is obtained        from the second micobacterium which has been cultured under        conditions that are not nutrient-starving and which support        exponential growth of said second, mycobacterium;    -   preparing first and second cDNA populations from said first- and        second mRNA populations respectively, during which cDNA        preparation a detectable label is introduced into the cDNA        molecules of the first and second cDNA populations;    -   isolating corresponding first and second cDNA molecules from the        first and second cDNA populations, respectively;    -   comparing relative amounts of label or corresponding signal        emitted from the label present in the isolated first and second        cDNA molecules;    -   identifying a greater amount of label or signal provided by the        isolated first cDNA molecule than that provided by the isolated        second cDNA molecule; and    -   identifying the first cDNA and the corresponding mycobacterial        gene which is induced or up-regulated during mycobacterial        latency.

Reference to gene throughout this specification embraces open readingframes (ORFs).

The various embodiments described for the first aspect of the presentinvention apply equally to the second and subsequent aspects of thepresent invention.

The term “corresponding first and second cDNA molecules from the firstand second cDNA populations” refers to cDNAs having substantially thesame nucleotide sequence. Thus, by isolating the cDNA copies relating toa given gene under each culture condition (ie. exponential phase, andstationary phase), it is possible to quantify the relative copy numberof cDNA for that gene for each culture condition. Since each cDNA copyhas been produced from an mRNA molecule, the cDNA copy number reflectsthe corresponding mRNA copy number for each culture condition, and thusit is possible to identify induced or up-regulated genes.

In one embodiment, the first and second cDNA molecules are isolated fromthe corresponding first and second cDNA populations by hybridization toan array containing immobilized DNA sequences that are representative ofeach known gene (or ORF) within a particular mycobacterial speciesgenome. Thus, a first cDNA may be considered “corresponding” to a secondcDNA if both cDNAs hybridize to the same immobilized DNA sequence.

In another embodiment, the first and second cDNAs are prepared byincorporation of a fluorescent label. The first and second cDNAs mayincorporate labels which fluoresce at different wavelengths, therebypermitting dual fluorescence and simultaneous detection of two cDNAsamples.

The type of label employed naturally determines how the output of thedetection method is read. When using fluorescent labels, a confocallaser scanner is preferably employed.

According to one embodiment, fluorescently labelled cDNA sequences fromstationary and exponential phase cultured systems were allowed tohybridize with a whole mycobacterial genome array. The first cDNApopulation was labelled with fluorescent label A, and the second cDNApopulation was labelled with fluorescent label B. The array was scannedat two different wavelengths corresponding to the excitable maxima ofeach dye and the intensity of the emitted light was recorded. Multiplearrays were preferably prepared for each cDNA and a mean intensity valuewas calculated across the two cDNA populations for each spot with eachdye, against which relative induction or upregulation was quantified.

In addition to the above mRNA isolation and cDNA preparation andlabelling, genomic DNA may be isolated from the first and secondmycobacteria. Thus, in a preferred embodiment, labelled DNA is alsoprepared from the isolated DNA. The labelled DNA may be then included oneach array as a control.

According to a third aspect of the present invention, there is providedan inhibitor of a mycobacterial peptide, wherein the peptide is encodedby a gene the expression of Which is induced or up-regulated underculture conditions that are nutrient-starving and which maintainmycobacterial latency, said conditions being obtainable by batchfermentation of a micobacterium for at least 20 days post-inoculation,when compared with culture conditions that are not nutrient-starving andwhich support exponential growth of said mycobacterium, wherein theinhibitor is capable of preventing or inhibiting the mycobacterialpeptide, from exerting its native biological effect.

Such inhibitors may be employed to prevent the onset of, or to cause abreak in the period of mycobacterial latency (ie. induce re-activation).In this respect, mycobacteria are more susceptible to treatment regimenswhen in a non-latent state, and the combined use of drugs to kill latentmycobacteria (eg. TB) would significantly reduce the incidence ofmycobacteria by targeting the reservoir for new disease and wouldthereby help reduce the problem of emerging drug-resistant strains.

The inhibitor may be a peptide, carbohydrate, synthetic molecule, or ananalogue thereof. Inhibition of the mycobacterial peptide may beeffected at the nucleic acid level (ie. DNA, or RNA), or at the peptidelevel. Thus, the inhibitor may act directly on the peptide.Alternatively, the inhibitor may act indirectly on the peptide by, forexample, causing inactivation of the induced or up-regulatedmycobacterial gene.

In preferred embodiments, the inhibitor is capable of inhibiting one ormore of the following:—2-nitropropane dioxygenase, acetyltransferase,oxidoreductase, transcriptional regulator, acyl transferase, UDP-glucosedehydrogenase, phosphoribosylglycinamide formyltransferase,1,4-dihydroxy-2-naphthoate octaprenyl, gmc-type oxidoreductase,3-hydroxyisobutyrate dehydrogenase, methylmalonate semialdehydedehydrogenase, dehydrogenase, mercuric reductase, glutathione reductase,dihydrolipoamide, transposase, proline iminopeptidase, protylaminopeptidase, quinolone efflux pump, glycine betaine transporter,phosphatidylethanolamine N-methyltransferase, chalcone synthase 2,sulfotransferase, glycosyl transferase, fumarate reductase-flavoprotein,8-amino-7-oxononanoate synthase, aminotransferase class-IIpyridoxal-phosphate, bacteriophage HK97 prohead protease,penicillin-binding protein, fatty acyl-CoA racemase, nitrilotriacetatemonooxygenase, histidine kinase response regulator, peptidase, LysRtranscription regulator, excisionase, ornithine aminotransferase, malateoxidoreductase, thiosulphate binding protein, enoyl-CoA hydratase,acyl-CoA synthetase, methyltransferase, siroheme synthase, permease,glutaryl 7-aca acylase, sn-glycerol-3-phosphate transport systempermease, enoyl-CoA hydratase/isomerase, acyl-CoA dehydrogenase,esterase, lipase, cytidine deaminase, crotonase, lipid-transfer protein,acetyl-CoA C-acetyltransferase, aminotransferase, hydrolase, and2-amino-4-hydroxy-6-hydroxymethyldihydropterine pyrophosphokinase.

In a further embodiment, the inhibitor may be an antibiotic capable oftargeting the induced or up-regulated mycobacterial gene identifiableby, the present invention, or the gene product thereof. The antibioticis preferably specific for the gene and/or gene product.

In a further embodiment, the inhibitor may act on a gene or gene productthe latter of which interacts with the induced or up-regulated gene.Alternatively, the inhibitor may act on a gene or gene product thereofupon which the gene product of the induced or up-regulated gene acts.

Inhibitors of the present invention may be prepared utilizing thesequence information of provided herein. For example, this may beperformed by overexpressing the peptide, purifying the peptide, and thenperforming X-ray crystallography on the purified peptide to obtain itsmolecular structure. Next, compounds are created which have similarmolecular structures to all or portions of the polypeptide or itssubstrate. The compounds may be then combined with the peptide andattached thereto so as to block one or more of its biologicalactivities.

Also included within the invention are isolated or recombinantpolynucleotides that bind to the regions of the mycobacterial chromosomecontaining sequences that are associated with induction/up-regulationunder low oxygen tension (ie. virulence), including antisense andtriplex-forming polynucleotides. As used herein, the term “binding”refers to an interaction or complexation between an oligonucleotide anda target nucleotide sequence, mediated through hydrogen bonding or othermolecular forces. The term “binding” more specifically refers to twotypes of internucleotide binding mediated through base-base hydrogenbonding. The first type of binding is “Watson-Crick-type” bindinginteractions in which adenine-thymine (or adenine-uracil) andguanine-cytosine base-pairs are formed through hydrogen bonding betweenthe bases. An example of this type of binding is the bindingtraditionally associated with the DNA double helix and in RNA-DNAhybrids; this type of binding is normally detected by hybridizationprocedures.

The second type of binding is “triplex binding”. In general, triplexbinding refers to any type of base-base hydrogen bonding of a thirdpolynucleotide strand with a duplex DNA (or DNA-RNA hybrid) that isalready paired in a Watson-Crick manner.

In a preferred embodiment, the inhibitor may be an antisense nucleicacid sequence which is complementary to at least part of the inducibleor up-regulatable gene.

The inhibitor, when in the form of a nucleic acid sequence, in use,comprises at least 15 nucleotides, preferably at least 20 nucleotides,more preferably at least 30 nucleotides, and most preferably at least 50nucleotides.

According to a fourth aspect of the invention, there is provided anantibody that binds to a peptide encoded by a gene, or to a fragment orvariant or derivative of said peptide, the expression of which gene isinduced or up-regulated during culture of a mycobacterium under cultureconditions that are nutrient-starving and which maintain mycobacteriallatency, said conditions being obtainable by batch fermentation of amycobacterium for at least 20 days post-inoculation, when compared withculture conditions that are not-nutrient-starving and which supportexponential growth of said mycobacterium.

The antibody preferably has specificity for the peptide in question, andfollowing binding thereto may initiate coating of the mycobacterium.Coating of the bacterium preferably leads to opsonization thereof. This,in turn, leads to the bacterium being destroyed. It is preferred thatthe antibody is specific for the mycobacterium (eg. species and/orstrain) which is to be targeted.

In use, the antibody is preferably embodied in an isolated form.

Opsonization by antibodies may influence cellular entry and spread ofmycobacteria in phagocytic and non-phagocytic cells by preventing ormodulating receptor-mediated entry and replication in macrophages.

The peptides, fragments, variants or derivatives of the presentinvention may be used to produce antibodies, including polyclonal andmonoclonal antibodies. If polyclonal antibodies are desired, a selectedmammal (eg. mouse, rabbit, goat, horse, etc.) is immunized with animmunogenic polypeptide. Serum from the immunized animal is collectedand treated according to known procedures. If serum containingpolyclonal antibodies to a desired mycobacterial epitope containsantibodies to other antigens, the polyclonal antibodies may be purifiedby immunoaffinity chromatography.

Alternatively, general methodology for making monoclonal antibodies byhybridomas involving, for example, preparation of immortalantibody-producing cell lines by cell fusion, or other techniques suchas direct transformation of B lymphocytes with oncogenic DNA, ortransfection with Epstein-Barr virus may be employed.

The antibody employed in this aspect of the invention may belong to anyantibody isotype family, or may be a derivative or mimic thereof.Reference to antibody throughout this specification embracesrecombinantly produced antibody, and any part of an antibody which iscapable of binding to a mycobacterial antigen.

In one embodiment the antibody belongs to the IgG, IgM or IgA isotypefamilies.

In a preferred embodiment, the antibody belongs to the IgA isotypefamily. Reference to the IgA isotype throughout this specificationincludes the secretory form of this antibody (ie. sIgA). The secretorycomponent (SC) of sIgA may be added in vitro or in vivo. In the lattercase, the use of a patient's natural SC labelling machinery may beemployed.

In one embodiment, the antibody may be raised against a peptide from amember of the MTC, preferably against M. tuberculosis.

In a preferred embodiment, the antibody is capable of binding to apeptide selected from the group consisting of SEQ ID NO: 1, 3, 5, 7, 9,11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81,83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113,115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169,171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197,199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225,227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253,255, 257, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281 anda fragment, variant, and derivative of said SEQ IDs.

In a further embodiment, the antigen is an exposed component of amycobacterial bacillus. In another embodiment, the antigen is, a cellsurface component of a mycobacterial bacillus.

The antibody of the present invention may be polyclonal, but ispreferably monoclonal.

Without being bound by any theory, it is possible that followingmycobacterial infection of a macrophage, the macrophage is killed andthe bacilli are released. It is at this stage that the mycobacteria areconsidered to be most vulnerable to antibody attack. Thus, it ispossible that the antibodies of the present invention act on releasedbacilli following macrophage death, and thereby exert a post-infectioneffect.

It is possible that the passive protection aspect (ie. delivery ofantibodies) of the present invention is facilitated by enhancedaccessibility of the antibodies of the present invention to antigens onmycobacterial bacilli harboured by the infected macrophages. Indeed, acrexpression is low during logarithmic growth, but increases at thestationary or oxygen limiting stage, and particularly in organisms whichreplicate within macrophages. As acr expression appears to be necessaryfor mycobacterial infectivity, it is possible that antibody binding mayblock macrophage infection by steric hindrance or disruption of itsoligomeric structure. Thus, antibodies acting on mycobacterial bacillireleased from killed, infected macrophages may interfere with the spreadof re-infection to fresh macrophages. This hypothesis involves asynergistic action between antibodies and cytotoxic T cells, actingearly after infection, eg. γδ and NK T cells, but could later involvealso CD8 and CD4 cytotoxic T cells.

According to a fifth aspect of the invention, there is provided anattenuated micobacterium in which a gene has been modified therebyrendering the micobacterium substantially non-pathogenic, wherein saidgene is a gene the expression of which is induced or up-regulated duringculture conditions that are nutrient-starving and which maintainmycobacterial latency, said conditions being obtainable by batchfermentation of a micobacterium for at least 20 days post-inoculation,when compared with culture conditions that are not nutrient-starving andwhich support exponential growth of said mycobacterium.

The modification preferably inactivates the gene in question, andpreferably renders the micobacterium substantially non-pathogenic.

The term “modified” refers to any genetic manipulation such as a nucleicacid or nucleic acid sequence replacement, a deletion, or an insertionwhich renders the micobacterium substantially reduced in ability topersist in a latent state. In one embodiment the entire inducible orup-regulatable gene may be deleted.

In a preferred embodiment, gene to be modified has a wild-type codingsequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44,46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80,82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112,114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196,198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224,226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250 252,254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280 and282.

It will be appreciated that the above wild-type sequences may includeminor variations depending on the Database employed. The term“wild-type” indicates that the sequence in question exists as a codingsequence in nature.

According to a sixth aspect of the invention, there is provided anattenuated microbial carrier, comprising a peptide encoded by a gene, ora fragment or variant or derivative of said peptide, the expression ofwhich gene is induced or up-regulated under culture conditions that arenutrient-starving and which maintain mycobacterial latency, saidconditions being obtainable by batch fermentation of a micobacterium forat least 20 days post-inoculation, when compared with culture conditionsthat are not nutrient-starving and which support exponential growth ofsaid mycobacterium.

In use, the peptide (or fragment, variant or derivative) is either atleast partially exposed at the surface of the carrier, or the carrierbecomes degraded in vivo so that at least part of the peptide (orfragment, variant or derivative) is otherwise exposed to a host's immunesystem.

In a preferred embodiment, the attenuated microbial carrier isattenuated salmonella, attenuated vaccinia virus, attenuated fowlpoxvirus, or attenuated M. bovis (eg. BCG strain).

In a preferred embodiment, the peptide is selected from the groupconsisting of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25,27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61,63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153,155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181,183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209,211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237,239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265,267, 269, 271, 273, 275, 277, 279 and 281.

According to a seventh aspect of the invention, there is provided a DNAplasmid comprising a promoter, a polyadenylation signal, and a DNAsequence that is the coding sequence of a mycobacterial gene or afragment or variant of derivative of said coding sequence, theexpression of which gene is induced or up-regulated under cultureconditions that are nutrient-starving and which maintain mycobacteriallatency, said conditions being obtainable by batch fermentation of a,micobacterium for at least 20 days post-inoculation, when compared withculture conditions that are not nutrient-starving and which supportexponential growth of said mycobacterium, wherein the promoter andpolyadenylation signal are operably linked to the DNA sequence.

The term DNA “fragment” used in this invention will usually comprise atleast about 5 codons (15 nucleotides), more usually at least about 7 to15 codons, and most preferably at least about 35 codons. This number ofnucleotides is usually about the minimal length required for asuccessful probe that would hybridize specifically with such a sequence.

In preferred embodiments, the DNA “fragment” has a nucleotide lengthwhich is at least 50%, preferably at least 70%, and more preferably atleast 80% that of the coding sequence of the correspondinginduced/up-regulated gene.

The term DNA “variant” means a DNA sequence that has substantialhomology or substantial similarity to the coding sequence (or a fragmentthereof) of an induced/up-regulated gene. A nucleic acid or fragmentthereof is “substantially homologous” (or “substantially similar”) toanother if, when optimally aligned (with appropriate nucleotideinsertions or deletions) with the other nucleic acid (or itscomplementary strand), there is nucleotide sequence identity in at leastabout 60% of the nucleotide bases, usually at least about 70%, moreusually at least about 80%, preferably at least about 90%, and morepreferably at least about 95 to 98% of the nucleotide bases. Homologydetermination is performed as described supra for peptides.

Alternatively, a DNA “variant” is substantially homologous (orsubstantially similar) with the coding sequence (or a fragment thereof)of an induced/up-regulated gene when they are capable of hybridizingunder selective hybridization conditions. Selectivity of hybridizationexists when hybridization occurs which is substantially more selectivethan total lack of specificity. Typically, selective hybridization willoccur when there is at least about 65% homology over a stretch of atleast about 14 nucleotides, preferably at least about 70%, morepreferably at least about 75%, and most preferably at least about 90%.See, Kanehisa (1984) Nuc. Acids Res. 12:203-213. The length of homologycomparison, as described, may be over longer stretches, and in certainembodiments will often be over a stretch of at least about 17nucleotides, usually at least about 20 nucleotides, more usually atleast about 24 nucleotides, typically at least about 28 nucleotides,more typically at least about 32 nucleotides, and preferably at leastabout 36 or more nucleotides.

Nucleic acid hybridization will be affected by such conditions as saltconcentration (eg. NaCl), temperature, or organic solvents, in additionto the base composition, length of the complementary strands, and thenumber of nucleotide base mismatches between the hybridizing nucleicacids, as will be readily appreciated by those skilled in the art.Stringent temperature conditions are preferably employed, and generallyinclude temperatures in excess of 30° C., typically in excess of 37° C.and preferably in excess of 45° C. Stringent salt conditions willordinarily be less than 1000 mM, typically less than 500 mM, andpreferably less than 200 mM. The pH is typically between 7.0 and 8.3.However, the combination of parameters is much more important than themeasure of any single parameter. See, eg., Wetmur and Davidson (1968) J.Mol. Biol. 31:349-370.

The term DNA “derivative” means a DNA polynucleotide which comprises aDNA sequence. (or a fragment, or variant thereof) corresponding to thecoding sequence of the induced/up-regulated gene and an additional DNAsequence which is not naturally associated with the DNA sequencecorresponding to the coding sequence. The comments on peptide derivativesupra also apply to DNA “derivative”. A “derivative” may, for example,include two or more coding sequences of a mycobacterial operon that isinduced during nutrient-starvation. Thus, depending on the presence orabsence of a non-coding region between the coding sequences, theexpression product/s of such a “derivative” may be a fusion protein, orseparate peptide products encoded by the individual coding regions.

The above terms DNA “fragment”, “variant”, and “derivative” have incommon with each other that the resulting peptide products havecross-reactive antigenic properties which are substantially the same asthose of the corresponding wild-type peptide. Preferably all of thepeptide products of the above DNA molecule embodiments of the presentinvention bind to an antibody which also binds to the wild-type peptide.Alternatively, all of the above peptide products are capable of inducinga “recall responses” of a T lymphocyte which has been previously exposedto an antigenic component of a mycobacterial infection.

The promoter and polyadenylation signal are preferably selected so as toensure that the, gene is expressed in a eukaryotic cell. Strongpromoters and polyadenylation signals are preferred.

In a related aspect, the present invention provides an isolated RNAmolecule which is encoded by a DNA sequence of the present invention, ora fragment or variant or derivative of said DNA sequence.

An “isolated” RNA is an RNA which is substantially separated from othermycobacterial components that naturally accompany the sequences ofinterest, eg., ribosomes, polymerases, and other mycobacterialpolynucleotides such as DNA and other chromosomal sequences.

The above RNA molecule may be introduced directly into a host cell as,for example, a component of a vaccine.

Alternatively the RNA molecule may be incorporated into an RNA vectorprior to administration.

The polynucleotide sequences (DNA and RNA) of the present inventioninclude a nucleic acid sequence which has been removed from itsnaturally occurring environment, and recombinant or cloned DNA isolatesand chemically synthesized analogues or analogues biologicallysynthesized by heterologous systems.

The term “recombinant” as used herein intends a polynucleotide ofgenomic, cDNA, semisynthetic, or synthetic origin which, by virtue ofits origin or manipulation: (1) is not associated with all or a portionof a polynucleotide with which it is associated in nature; or (2) islinked to a polynucleotide other than that to which it is linked innature; and (3) does not occur in nature. This artificial combination isoften accomplished by either chemical synthesis means, or by theartificial manipulation of isolated segments of nucleic acids, eg., bygenetic engineering techniques. Such is usually done to replace a codonwith a redundant codon encoding the same or a conservative amino acid,while typically introducing or removing a sequence recognition site.Alternatively, it is performed to join together nucleic acid segments ofdesired functions to generate a desired combination of functions.

In embodiments of the invention the polynucleotides may encode a peptide(or fragment, variant, or derivative) which is induced or up-regulatedunder nutrient-starving conditions. A nucleic acid is said to “encode” apeptide if, in its native state or when manipulated, it can betranscribed and/or translated to produce the peptide (or fragment,variant or derivative thereof). The anti-sense strand of such a nucleicacid is also said to encode the peptide (or fragment, variant, orderivative).

Also contemplated within the invention are expression vectors comprisingthe polynucleotide of interest. Expression vectors “generally” arereplicable polynucleotide constructs that encode a peptide operablylinked to suitable transcriptional and translational regulatoryelements. Examples of regulatory elements usually included in expressionvectors are promoters enhancers, ribosomal binding sites, andtranscription and translation initiation and termination sequences.These regulatory elements are operably linked to the sequence to betranslated. A nucleic acid sequence is operably linked when it is placedinto a functional relationship with another nucleic acid sequence. Forinstance, a promoter is operably linked to a coding sequence if thepromoter affects its transcription or expression. Generally, operablylinked means that the DNA sequences being linked are contiguous and,where necessary to join two protein coding regions, contiguous and inreading frame. The regulatory elements employed in the expressionvectors containing a polynucleotide encoding a virulence factor arefunctional in the host cell used for expression.

The polynucleotides of the present invention may be prepared by anymeans known in the art. For example, large amounts of thepolynucleotides may be produced by replication in a suitable host cell.The natural or synthetic DNA fragments coding for a desired fragmentwill be incorporated into recombinant nucleic acid constructs, typicallyDNA constructs, capable of introduction into and replication in aprokaryotic or eukaryotic cell. Usually the DNA constructs will besuitable for autonomous replication in a unicellular host, such as yeastor bacteria, but may also be intended for introduction to andintegration within the genome of a cultured insect, mammalian, plant orother eukaryotic cell lines.

The polynucleotides of the present invention may also be produced bychemical synthesis, e.g., by the phosphoramidite method or the triestermethod, and may be performed on commercial automated oligonucleotidesynthesizers. A double-stranded fragment may be obtained from the singlestranded product of chemical synthesis either by synthesizing thecomplementary strand and annealing the strand together under appropriateconditions or by adding the complementary strand using DNA polymerasewith an appropriate primer sequence.

DNA constructs prepared for introduction into a prokaryotic oreukaryotic host will typically comprise a replication system recognizedby the host, including the intended DNA fragment encoding the desiredpeptide, and will preferably also include transcription andtranslational initiation regulatory sequences operably linked to thepolypeptide encoding segment. Expression vectors may include, forexample, an origin of replication or autonomously replicating sequence(ARS) and expression control sequences, a promoter, an enhancer andnecessary processing information sites, such as ribosome binding sites,RNA splice sites, polyadenylation sites, transcriptional terminatorsequences, and mRNA stabilizing sequences. Secretion signals frompolypeptides secreted from the host cell of choice may also be includedwhere appropriate, thus allowing the protein to cross and/or lodge incell membranes, and thus attain its functional topology or be secretedfrom the cell.

Appropriate promoter and other necessary vector sequences will beselected so as to be functional in the host, and may, when appropriate,include those naturally associated with mycobacterial genes. Promoterssuch as the trp, lac and phage promoters, tRNA promoters and glycolyticenzyme promoters may be used in prokaryotic hosts. Useful yeastpromoters include the promoter regions for metallothionein,3-phosphoglycerate kinase or other glycolytic enzymes such as enolase orglyceraldehyde-3-phosphate dehydrogenase, enzymes responsible formaltose and galactose utilization, and others.

Appropriate non-native mammalian promoters may include the early andlate promoters from SV40 or promoters derived from murine moloneyleukemia virus, mouse mammary tumour virus, avian sarcoma viruses,adenovirus II, bovine papilloma virus or polyoma. In addition, theconstruct may be joined to an amplifiable-gene (e.g., DHFR) so thatmultiple copies of the gene may be made

While such expression vectors may replicate autonomously, they may lesspreferably replicate by being inserted into the genome of the host cell.

Expression and cloning vectors will likely contain a selectable marker,a gene encoding a protein necessary for the survival or growth of a hostcell transformed with the vector. The presence of this gene ensures thegrowth of only those host cells which express the inserts. Typicalselection genes encode proteins that (a) confer resistance toantibiotics or other toxic substances, e.g. ampicillin, neomycin,methotrexate, etc.; (b) complement auxotrophic deficiencies; or (c)supply critical nutrients not available from complex media, e.g. thegene encoding D-alanine racemase for Bacilli. The choice of appropriateselectable marker will depend on the host cell.

The vectors containing the nucleic acids of interest can be transcribedin vitro and the resulting RNA introduced into the host cell (e.g., byinjection), or the vectors can be introduced directly into host cells bymethods which vary depending on the type of cellular host, includingelectroporation; transfection employing calcium chloride, rubidiumchloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; infection (where the vector isan infectious agent, such as a retroviral genome). The cells into whichhave been introduced nucleic acids described above are meant to alsoinclude the progeny of such cells.

Large quantities of the nucleic acids and peptides of the presentinvention may be prepared by expressing the nucleic acids or portionsthereof in vectors or other expression vehicles in compatibleprokaryotic or eukaryotic host cells. The most commonly used prokaryotichosts are strains of Escherichia coli, although other prokaryotes, suchas Bacillus subtilis or Pseudomonas may also be used.

Mammalian or other eukaryotic host cells, such as those of yeast,filamentous fungi, plant, insect, amphibian or avian species, may alsobe useful for production of the proteins of the present invention.Propagation of mammalian cells in culture is per se well known. Examplesof commonly used mammalian host cell lines are VERO and HeLa cells,Chinese hamster ovary (CHO) cells, and WI38, BHK, and COS cell lines,although other cell lines may be appropriate, e.g., to provide higherexpression, desirable glycosylation patterns.

Clones are selected by using markers depending on the mode of the vectorconstruction. The marker may be on the same or a different DNA molecule,preferably the same DNA molecule. The transformant may be screened or,preferably, selected by any of the means well known in the art, e.g., byresistance to such antibiotics as ampicillin, tetracycline.

The polynucleotides of the invention may be inserted into the host cellby any means known in the art, including for example, transformation,transduction, and electroporation. As used herein, “recombinant hostcells”, “host cells”, “cells”, “cell lines”, “cell cultures”, and othersuch terms denoting microorganisms or higher eukaryotic cell linescultured as unicellular entities refer to cells which can be, or havebeen, used as recipients for recombinant vector or other transfer DNA,and include the progeny of the original cell which has been transformed.It is understood that the progeny of a single parental cell may notnecessarily be completely identical in morphology or in genomic or totalDNA complement as the original parent, due to natural, accidental, ordeliberate mutation. “Transformation”, as used herein, refers to theinsertion of an exogenous polynucleotide into a host cell, irrespectiveof the method used for the insertion, for example, direct uptake,transduction, f-mating or electroporation. The exogenous polynucleotidemay be maintained as a non-integrated vector, for example, a plasmid, oralternatively, may be integrated into the host cell genome.

In one embodiment, a DNA plasmid or RNA vector may encode a component ofthe immune system which is specific to an immune response followingchallenge with a peptide, wherein said peptide is encoded by amycobacterial gene that is induced or up-regulated duringnutrient-starvation, and optionally oxygen starvation.

An example of such a component is an antibody to the peptide product ofthe induced or up-regulated gene. Thus, in one embodiment, the nucleicacid sequence (eg. DNA plasmid, or RNA vector) encodes the antibody inquestion.

An eighth aspect provides use of the aforementioned aspects of thepresent invention, namely a peptide or fragment or variant or derivativethereof, an inhibitor, an antibody, an attenuated mycobacterium, anattenuated microbial carrier, a DNA sequence that is the coding sequenceof an induced or up-regulated mycobacterial gene or a fragment orvariant or derivative of said coding sequence, a DNA plasmid comprisingsaid DNA sequence, an RNA sequence encoded by said DNA sequence(including DNA fragment, variant, derivative), and/or an RNA vectorcomprising said RNA, sequence, in the manufacture of a medicament fortreating or preventing a mycobacterial infection.

The term “preventing” includes reducing the severity/intensity of, orinitiation of, a mycobacterial infection.

The term “treating” includes post-infection therapy and amelioration ofa mycobacterial infection.

In a related aspect, there is provided a method of treating orpreventing a mycobacterial infection, comprising administration of amedicament (namely the aforementioned aspects of the present invention)selected from the group consisting of a peptide or fragment or variantor derivative thereof, an inhibitor, an antibody, an attenuatedmycobacterium, an attenuated microbial carrier, a DNA sequence that isthe coding sequence of an induced or up-regulated mycobacterial gene ora fragment or variant or derivative of said coding sequence, a DNAplasmid comprising said DNA sequence, an RNA sequence encoded by saidDNA sequence, and/or an RNA vector comprising said RNA sequence, to apatient.

The immunogenicity of the epitopes of the peptides of the invention maybe enhanced by preparing them in mammalian or yeast systems fused withor assembled with particle-forming proteins such as, for example, thatassociated with hepatitis B surface antigen. Vaccines may be preparedfrom one or more immunogenic peptides of the present invention.

Typically, such vaccines are prepared as injectables, either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid prior to injection may also be prepared. Thepreparation may also be emulsified, or the peptide encapsulated inliposomes. The active immunogenic ingredients are often mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the vaccine may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,and/or adjuvants which enhance the effectiveness of the vaccine.Examples of adjuvants which may be effective include but are not limitedto: aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine(thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637,referred to as nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion.

The vaccines are conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations orformulations suitable for distribution as aerosols. For suppositories,traditional binders and carriers may include, for example, polyalkyleneglycols or triglycerides; such suppositories may be formed from mixturescontaining the active ingredient in the range of 0.5% to 10%, preferably1%-2%. Oral formulations include such normally employed excipients as,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,and the like. These compositions take the form of solutions,suspensions, tablets, pills, capsules, sustained release formulations orpowders and contain 10%-95% of active ingredient, preferably 25%-70%.

The peptides may be formulated into the vaccine as neutral or saltforms. Pharmaceutically acceptable salts include the acid addition salts(formed with free amino groups of the peptide) and which are formed withinorganic acids such as, for example, hydrochloric or phosphoric acids,or with organic acids such as acetic, oxalic, tartaric, maleic, and thelike. Salts formed with the free carboxyl groups may also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be prophylactically and/ortherapeutically effective. The quantity to be administered, which isgenerally in the range of 5 micrograms to 250 micrograms of antigen perdose, depends on the subject to be treated, capacity of the subject'simmune, system to synthesize antibodies, and the degree of protectiondesired. Precise amounts of active ingredient required to beadministered may depend on the judgment of the practitioner and may bepeculiar to each subject.

The vaccine may be given in a single dose schedule, or preferably in amultiple dose schedule. A multiple dose schedule is one in which aprimary course of vaccination may be with 1-10 separate doses, followedby other doses given at subsequent time intervals required to maintainand or re-enforce the immune response, for example, at 1-4 months for asecond dose, and if needed, a subsequent dose(s) after several months.The dosage-regimen will also, at least in part, be determined by theneed of the individual and be dependent upon the judgment of thepractitioner.

In addition, the vaccine containing the immunogenic mycobacterialantigen(s) may be administered in conjunction with otherimmunoregulatory agents; for example, immunoglobulins, as well asantibiotics.

The medicament may be administered by conventional routes, eg.intravenous, intraperitoneal, intranasal routes.

The outcome of administering antibody-containing compositions may dependon the efficiency of transmission of antibodies to the site ofinfection. In the case of a mycobacterial respiratory infection (eg. aM. tuberculosis infection), this may be facilitated by efficienttransmission of antibodies to the lungs.

In one embodiment the medicament may be administered intranasally(i.n.). This mode of delivery corresponds to the route of delivery of aM. tuberculosis infection and, in the case of antibody delivery, ensuresthat antibodies are present at the site of infection to combat thebacterium before it becomes intracellular and also during the periodwhen it spreads between cells.

An intranasal composition may be administered in droplet form havingapproximate diameters in the range of 100-5000 μm, preferably 500-4000μm; more preferably 1000-3000 μm. Alternatively, in terms of volume, thedroplets would be in the approximate range of 0.001-100 μl, preferably0.1-50 μl, more preferably 1.0-25 μl.

Intranasal administration may be achieved by way of applying nasaldroplets or via a nasal spray.

In the case of nasal droplets, the droplets may typically have adiameter of approximately 1000-3000 μm and/or a volume of 1-25 μl.

In the case of a nasal spray, the droplets may typically have a diameterof approximately 100-1000 μm and/or a volume of 0.001-1 μl.

It is possible that, following i.n. delivery of antibodies, theirpassage to the lungs is facilitated by a reverse flow of mucosalsecretions, although mucociliary action in the respiratory tract isthought to take particles within the mucus out of the lungs. Therelatively long persistence in the lungs' lavage, fast clearance fromthe bile and lack of transport to the saliva of some antibodies suggestthe role of mucosal site specific mechanisms.

In a different embodiment, the medicament may be delivered in an aerosolformulation. The aerosol formulation may take the form of a powder,suspension, or solution.

The size of aerosol particles is one factor relevant to the deliverycapability of an aerosol. Thus, smaller particles may travel furtherdown the respiratory airway towards the alveoli than would largerparticles. In one embodiment, the aerosol particles have a diameterdistribution to facilitate delivery along the entire length of thebronchi, bronchioles, and alveoli. Alternatively, the particle sizedistribution may be selected to target a particular section of therespiratory airway, for example the alveoli.

The aerosol particles may be delivered by way of a nebulizer or nasalspray.

In the case of aerosol delivery of the medicament, the particles mayhave diameters in the approximate range of 0.1-50 μm, preferably 1-25μm, more preferably 1-5 μm.

The aerosol formulation of the medicament of the present invention mayoptionally contain a propellant and/or surfactant.

By controlling the size of the droplets which are to be administered toa patient to within the defined range of the present invention, it ispossible to avoid/minimise inadvertent antigen delivery to the alveoliand thus avoid alveoli-associated pathological problems such asinflammation and fibrotic scarring of the lungs.

I.n. vaccination engages both T and B cell mediated effector mechanismsin nasal and bronchus associated mucosal tissues, which differ fromother mucosae-associated lymphoid tissues.

The protective mechanisms invoked by the intranasal route ofadministration may include: the activation of T lymphocytes withpreferential lung homing; upregulation of co-stimulatory molecules, eg.B7.2; and/or activation of macrophages or secretory IgA antibodies.

Intranasal delivery of antigens may facilitate a mucosal antibodyresponse is involved which is favored by a shift in the T cell responsetoward the Th2 phenotype which helps antibody production. A mucosalresponse is characterized by enhanced IgA production, and a Th2 responseis characterized by enhanced IL-4 production.

Intranasal delivery of mycobacterial antigens allows targeting of theantigens to submucosal B cells of the respiratory system. These B cellsare the major local IgA-producing cells in mammals and intranasaldelivery facilitates a rapid increase in IgA production by these cellsagainst the mycobacterial antigens.

In one embodiment administration of the medicament comprising amycobacterial antigen stimulates IgA antibody production, and the IgAantibody binds to the mycobacterial antigen. In another embodiment, amucosal and/or Th2 immune response is stimulated.

In another embodiment monoclonal antibodies, in particular, may be usedto raise anti-idiotype antibodies. Anti-idiotype antibodies areimmunoglobulins which carry an “internal image” of the antigen of theinfectious agent against which protection is desired. Theseanti-idiotype antibodies may also be useful for treatment, vaccinationand/or diagnosis of mycobacterial infections.

According to a ninth aspect of the present invention, the peptides(including fragments, variants, and derivatives thereof) of the presentinvention and antibodies which bind thereto are useful in immunoassaysto detect the presence of antibodies to mycobacteria, or the presence ofthe virulence associated antigens in biological samples. Design of theimmunoassays is subject to a great deal of variation, and many formatsare known in the art. The immunoassay may utilize at least one epitopederived from a peptide of the present invention. In one embodiment, theimmunoassay uses a combination of such epitopes. These epitopes may bederived from the same or from different bacterial peptides, and may bein separate recombinant or natural peptides, or together in the samerecombinant peptides.

An immunoassay may use, for example, a monoclonal antibody directedtowards a virulence associated peptide epitope(s), a combination ofmonoclonal antibodies directed towards epitopes of one mycobacterialantigen, monoclonal antibodies directed towards epitopes of differentmycobacterial antigens, polyclonal antibodies directed towards the sameantigen, or polyclonal antibodies directed towards different antigens.Protocols may be based, for example, upon competition, or directreaction, or sandwich type assays. Protocols may also, for example, usesolid supports, or may be by immunoprecipitation. Most assays involvethe use of labelled antibody for polypeptide; the labels may be, forexample, enzymatic, fluorescent, chemiluminescent, radioactive, or dyemolecules. Assays which amplify the signals from the probe are alsoknown; examples of which are assays which utilize biotin and avidin, andenzyme-labelled and mediated immunoassays, such as ELISA assays.

Typically, an immunoassay for an antibody(s) to a peptide, will involveselecting and preparing the test sample suspected of containing theantibodies, such as a biological sample, then incubating it with anantigenic (i.e., epitope-containing) peptide(s) under conditions thatallow antigen-antibody complexes to form, and then detecting theformation of such complexes. The immunoassay may be of a standard orcompetitive type.

The peptide is typically bound to a solid support to facilitateseparation of the sample from the peptide after incubation. Examples ofsolid supports that can be used are nitrocellulose (e.g., in membrane ormicrotiter well form), polyvinyl chloride (e.g., in sheets or microtiterwells), polystyrene latex (e.g. in beads or microtiter plates,polyvinylidine fluoride (known as Immulon), diazotized paper, nylonmembranes, activated beads, and Protein A beads. For example, DynatechImmulon microtiter plates or 60 mm diameter polystyrene beads (PrecisionPlastic Ball) may be used. The solid support containing the antigenicpeptide is typically washed after separating it from the test sample,and prior to detection of bound antibodies.

Complexes formed comprising antibody (or, in the case of competitiveassays, the amount of competing antibody) are detected by any of anumber of known techniques, depending on the format. For example,unlabelled antibodies in the complex may be detected using a conjugateof antixenogeneic Ig complexed with a label, (e.g., an enzyme label).

In immunoassays where the peptides are the analyte, the test sample,typically a biological sample, is incubated with antibodies directedagainst the peptide under conditions that allow the formation ofantigen-antibody complexes. It may be desirable to treat the biologicalsample to release putative bacterial components prior to testing.Various formats can be employed. For example, a “sandwich assay” may beemployed, where antibody bound to a solid support is incubated with thetest sample; washed; incubated with a second, labelled antibody to theanalyte, and the support is washed again. Analyte is detected bydetermining if the second antibody is bound to the support. In acompetitive format, a test sample is usually incubated with antibody anda labelled, competing antigen is also incubated, either sequentially orsimultaneously.

Also included as an embodiment of the invention is an immunoassay kitcomprised of one or more peptides of the invention, or one or moreantibodies to said peptides, and a buffer, packaged in suitablecontainers.

As used herein, a “biological sample” refers to a sample of tissue orfluid isolated from an individual, including but not limited to, forexample, plasma, serum, spinal fluid, lymph fluid, the external sectionsof the skin, respiratory, intestinal and genitourinary tracts, tears,saliva, milk, blood cells, tumours, organs, and also samples of in vitrocell culture constituents (including but not limited to conditionedmedium resulting from the growth of cells in cell culture medium,putatively virally infected cells, recombinant cells, and cellcomponents).

In a related diagnostic assay, the present invention provides nucleicacid probes or detecting a mycobacterial infection.

Using the polynucleotides of the present invention as a basis, oligomersof approximately 8 nucleotides or more can be prepared, either byexcision from recombinant polynucleotides or synthetically, whichhybridize with the mycobacterial sequences, and are useful inidentification of mycobacteria. The probes are a length which allows thedetection of the induced or up-regulated sequences by hybridization.While 6-8 nucleotides may be a workable length, sequences of 10-12nucleotides are preferred, and at least about 20 nucleotides appearsoptimal. These probes can be prepared using routine methods, includingautomated oligonucleotide synthetic methods. For use as probes, completecomplementarity is desirable, though it may be unnecessary as the lengthof the fragment is increased.

For use of such probes as diagnostics, the biological sample to beanalyzed, such as blood or serum, may be treated, if desired, to extractthe nucleic acids contained therein. The resulting nucleic acid from thesample may be subjected to gel electrophoresis or other size separationtechniques; alternatively, the nucleic acid sample may be dot blottedwithout size separation. The probes are usually labeled. Suitablelabels, and methods for labeling probes are known in the art, andinclude, for example, radioactive labels incorporated by nicktranslation or kinasing, biotin, fluorescent probes, andchemiluminescent probes. The nucleic acids extracted from the sample arethen treated with the labeled probe under hybridization conditions ofsuitable stringencies.

The probes may be made completely complementary to the virulenceencoding polynucleotide. Therefore, usually high stringency conditionsare desirable in order to prevent false positives. The stringency ofhybridization is determined by a number of factors during hybridizationand during the washing procedure, including temperature, ionic strength,length of time, and concentration of formamide.

It may be desirable to use amplification techniques in hybridizationassays. Such techniques are known in the art and include, for example,the polymerase chain reaction (PCR) technique.

The probes may be packaged into diagnostic kits. Diagnostic kits includethe probe DNA, which may be labelled; alternatively, the probe DNA maybe unlabeled and the ingredients for labelling may be included in thekit in separate containers. The kit may also contain other suitablypackaged reagents and materials needed for, the particular hybridizationprotocol, for example, standards, as well as instructions for conductingthe test.

In a preferred embodiment, a peptide (or fragment or variant orderivative) of the present invention is used in a diagnostic assay todetect the presence of a T-lymphocyte which T lymphocyte has beenpreviously exposed to an antigenic component of a mycobacterialinfection in a patient.

In more detail, a T-lymphocyte which has been previously exposed to aparticular antigen will be activated on subsequent challenge by the sameantigen. This activation provides a means for identifying a positivediagnosis of mycobacterial infection. In contrast, the same activationis not achieved by a T-lymphocyte which has not been previously exposedto the particular antigen.

The above “activation” of a T-lymphocyte is sometimes referred to as a“recall response” and may be measured, for example, by determining therelease of interferon (eg. IFN-Y) from the activated T-lymphocyte. Thus,the presence of a mycobacterial infection in a patient may be determinedby the release of a minimum concentration of interferon from aT-lymphocyte after a defined time period following in vitro challenge ofthe T-lymphocyte with a peptide (or fragment or variant or derivative)of the present invention.

In use, a biological sample containing T-lymphocytes is taken from apatient, and then challenged with a peptide (or fragment, variant, Orderivative thereof) of the present invention.

The above T-lymphocyte diagnostic assay may include an antigenpresenting cell (APC) expressing at least one major histocompatibilitycomplex (MHC) class II molecule expressed by the patient in question.The APC may be inherently provided in the biological sample, or may beadded exogenously. In one embodiment, the T-lymphocyte is a CD4T-lymphocyte.

Brief mention is now made to the Figures of the present application, inwhich:—

FIG. 1 illustrates the viable counts for M. tuberculosis during cultureunder batch fermentation conditions at a DOT of 50% air saturation (37°C.); and

FIG. 2 illustrates the concentration of glycerol (as the primary carbonand energy source during culture of M. tuberculosis under batchfermentation conditions at a DOT of 50% air saturation (37° C.).

FIG. 3 illustrates the DOT within the medium of the mycobacterialculture described in Example 18.

FIG. 4 illustrates the viable counts for M. tuberculosis during thebatch fermentation conditions of Example 18 (ie. carbon-starvation, andoxygen limiting conditions).

EXAMPLE 1 Culture of Mycobacteria

Two alternative mycobacterial culture methods have been employed tostudy genes which are up-regulated or induced during mycobacteriallatency.

-   Model 1—In vitro Model of Mycobacterial Persistence Under Aerobic,    Nutrient-starved Conditions    Materials and Methods    Strain

Studies were performed with M. tuberculosis strain H37Rv (NCTC cat. no.7416)—a representative strain of M. tuberculosis. Stock cultures weregrown on Middlebrook 7H10+OADC for 3 weeks at 37±2° C.

Culture Medium

Persistence cultures were established in Middlebrook 7H9 mediumsupplemented with Middlebrook ADC enrichment, 0.2% Tween 80 and 0.2%glycerol (Table 1). The medium was prepared with high quality water froma Millipore water purification system and filter sterilized by passagethrough a 0.1 μm pore size cellulose acetate membrane filter capsule(Sartorius Ltd). The pH was adjusted to 6.6 with concentratedhydrochloric acid.

Middlebrook 7H10+OADC agar was used to prepare inoculum cultures,enumerate the number of culturable bacteria in samples, and to assessculture purity.

Culture System

We previously developed a process for the culture of mycobacteria undercontrolled and defined conditions—patent application No. PCT/GB00/00760(WO00/52139). We used this culture system operated as a batch fermenterfor the following studies of mycobacterial persistence.

Culture experiments were performed in a one liter glass vessel operatedat a working volume of 750 ml. The culture was agitated by a magneticbar placed in the culture vessel coupled to a magnetic stirrerpositioned beneath the vessel. Culture conditions were continuouslymonitored by an Anglicon Microlab Fermentation System (Brighton Systems,Newhaven), linked to sensor probes inserted into the culture throughsealed ports in the top plate. The oxygen concentration was monitoredwith a galvanic oxygen electrode (Uniprobe, Cardiff) and was controlledthrough sparging the culture with a mixture of air and oxygenfree-nitrogen. Temperature was monitored by an Anglicon temperatureprobe, and maintained by a heating pad positioned beneath the culturevessel. Culture pH was measured using an Ingold pH electrode(Mettler-Toledo, Leicester).

Inoculation and Culture

The vessel was filled with 750 ml of sterile culture medium andparameters were allowed to stabilise stabilize at 37° C.±2° C., pH6.9±0.3 and a dissolved oxygen tension of approximately 70% airsaturation. A dense inoculum suspension was prepared by resuspendingMiddlebrook agar cultures, grown at 37±2° C. for 3 weeks, in steriledeionized water. The inoculum was aseptically transferred to the culturevessel, to provide an initial culture turbidity of approximately 0.25 at540 nm.

The culture were maintained at 37° C. with an agitation rate of 500 to750 rpm. The dissolved oxygen tension was maintained between 50-70% airsaturation with the aid of culture sparging. The initial culture pH wasset at approximately 6.7 and was monitored through-out the experiment.

The culture was maintained for 50 days and samples were removedregularly to monitor growth and survival, nutrient utilization and geneexpression.

Growth and Survival.

Bacterial growth and survival was assessed by determining the number ofviable cells in the culture system at specific time points. This wasachieved by preparing a decimal dilution series of the sample in sterilewater and plating 100 μl aliquots onto Middlebrook 7H10+OADC plates. Theplates were incubated at 37° C. for up to 4 weeks before enumerating thenumber of colonies formed.

Nutrient Utilization

Glycerol is the primary carbon and energy source present in Middlebrook7H9 medium with ADC, 0.2% Tween and 0.2% Glycerol. The rate at whichglycerol was utilized was determined using the Glycerol DeterminationKit Cat. No. 148 270 Boehringer Manheim.

Microarray Experiments

RNA was extracted from culture samples collected at different timepoints during the experiment. A fluorescently-labelled cDNA was thentranscribed from each sample of RNA. The cDNA was labelled by theincorporation of either Cy3 or Cy5 labelled dCTP (Dyes are supplied byAmersham Pharmacia Biotech).

Whole M. tuberculosis genome arrays were prepared from M. tuberculosisgenomic DNA using ORF-specific primers. PCR products corresponding toeach ORF were spotted in a grid onto a standard glass microscope slideusing a BioRobotics microgrid robot (MWG Biotech) at a resolutionof >4000 spots/cm².

In each microarray experiment a whole genome array was hybridized withlabelled cDNA from one culture sample (Test sample). Each array was alsohybridized with control DNA incorporating a different Cy dye andprepared from DNA extracted from M. tuberculosis strain H37Rv (controlsample).

Each array was scanned at two different wavelengths corresponding to theexcitation maxima of each dye and the intensity of the emitted light wasrecorded. The ration of the intensity values for the test and controlsamples was determined for each array.

The slides were scanned using an Affymetrix 428 scanner. The raw datawas initially analyzed by ImaGene software. The scanned images were thentransferred to another software package known as GeneSpring to analyzethe expression of each gene.

Results

After inoculation the culture entered exponential growth and continuedto grow exponentially until 10 days after inoculation (see FIG. 1).Cessation of exponential growth coincided with depletion of the primarycarbon and energy source—glycerol (see FIG. 2). As the culture enteredstationary phase, viability started to decline and continued to declinesteadily over the duration of the study. After 40 days in stationaryphase, approximately 1% of the culture was still culturable onMiddlebrook agar.

The gene expression profiles for samples collect at day 5 and day 50were compared. Three arrays were prepared for each sample and the ratioof the intensity values for the test and control samples was determinedfor each array.

Two different approaches were used to analyze the data:—

-   -   1. The ratio values for the 3 arrays prepared for: each sample        were averaged and compared. Genes which produced intensity        ratios that were 3-fold higher on day 50 than on day 5 were        selected.    -   2. Data from each array was treated as a separate data set and        self-organizing maps were used to select all the genes that were        consistently up-regulated in all 3 arrays at day 50 relative to        day 5.

The two data sets were then compared and those genes that were at least1.5-fold, preferably at least 3-fold up-regulated at day 50, relative toexponential growth at day 5, and which were consistently up-regulated inall 3 arrays (experiments) were selected. The identified sequences(protein, followed by nucleic acid) are presented in Table 2.

-   Model 2—In vitro Model of Mycobacterial Persistence Under Low    Oxygen, and Nutrient-starved Conditions

A second model which simulated low-oxygen availability and nutrientdepletion has also been developed. This model was established asoutlined for Model 1 above, but with the following modifications.

After inoculation, the dissolved oxygen tension (DOT) of the culture wasmaintained at approximately 40% air saturation at 37° C. until theculture had entered early exponential growth. The DOT was then loweredin increments down to 1% air saturation over a six day period. Theculture was then maintained at a DOT of 0-5% until 50 days afterinoculation. Samples were collected for analysis, and the identifiedsequences (protein, followed by nucleic acid) are presented in Table 2.

TABLE 1 liquid medium formulation for persistence cultures - Middlebrook7H9 medium supplemented with ADC, 0.2% Tween 80 and 0.2% GlycerolComposition per liter Na₂HPO₄ 2.5 g KH₂PO₄ 1.0 g Monosodium glutamate0.5 g (NH₄)₂SO₄ 0.5 g Sodium citrate 0.1 g MgSO₄.7H₂O 0.05 g Ferricammonium citrate 0.04 g CuSO₄.5H₂O 1.0 mg Pyridoxine 1.0 mg ZnSO₄.7H₂O1.0 mg Biotin 0.5 mg CaCl₂.2H₂O 0.5 mg Middlebrook ADC enrichment 100 mlGlycerol 2.0 ml Tween 80 2.0 ml Middlebrook ADC enrichment - per 100 mlBovine serum albumin 5.0 g Glucose 2.0 g Catalase 3.0 mg

EXAMPLE 2 RNA Extraction from M. tuberculosis for Microarray Analysis

Materials and Methods

Trizol (Life Technologies)—Formulation of Phenol and GuanidineThiocyanate.

GTC lysis solution containing: 5 M guanidine thiocyanate, 0.5% N-laurylsarcosine, 25 mM tri-sodium citrate, 0.1 M 2-mercaptoethanol, and 0.5%Tween 80.

-   Chloroform-   Isopropanol-   3M sodium acetate-   70% Ethanol-   microfuge-   ribolyser-   Sterile plasticware-Falcon tubes, screw capped eppendorfs, gilson    tips—all RNase free-   Glassware—baked at 160° C. for at least 16 hours    Method

Steps performed at Containment level 3; within a Class IIImicrobiological safety cabinet.

Remove 10 or 20 ml of culture (10⁹/ml) and immediately add this to 4volumes of GTC lysis buffer in a plastic specimen pot. Seal the pottightly.

Incubate the cells in GTC lysis buffer for 1 hour at room temperature.Surface decontaminate the plastic pot with 5% Hycolin for 5 minutes.Transfer the sample to the pass box and place it into a plastic carrytin with a sealable lid. Close the container securely and transport itto a non-toxic cabinet CL3 cabinet.

Equally distribute the lysis mixture between Falcon tubes. Place thesetubes into centrifuge buckets and seal the buckets tightly.Surface-decontaminate the buckets for 5 minutes with 5% Hycolin. Thentransfer them to the centrifuge (Baird and Tatlock Mark IV refrigeratedbench-top centrifuge). Spin the tubes at 3,000 rpm for 30 minutes.

Return the unopened buckets to the cabinet. Remove the centrifuge tubesand pour the supernatant into a waste bottle for GTC lysis buffer.

Resuspend each pellet in 1 ml of Trizol (formulation of phenol and GTCcat no. 15596-026). The manufacturers guidelines recommend lysing cellsby repetitive pipetting. Although this action alone will not lyse M.tuberculosis, it is important to completely resuspend the pellet inTrizol.

Transfer 1 ml of cells into a FastRNA tube and ribolyse it at powersetting 6.5 for 45 seconds.

Leave the tube to incubate at room temperature for 5 minutes.

Remove the aqueous layer from the tube and add this to 200 μl ofchloroform in a screw-capped eppendorf tube. Shake each tube vigorouslyfor about 15 seconds. Incubate for 2-3 minutes at room temperature.

Spin the tube at 13,000 rpm for 15 minutes. Following centrifugation,the liquid separates into red phenol/chloroform phase, an interface, anda clear aqueous phase.

Carefully remove the aqueous phase and transfer it to a fresh eppendorftube containing 500 μl of chloroform/isoamyl alcohol (24:1). Spin thetubes at 13,000 rpm for 15 minutes.

Transfer the aqueous phase to an eppendorf tube containing 50 μl ofsodium acetate and 500 μl of isopropanol.

Surface decontaminate the eppendorf tube with 5% Hycolin for 5 minutes.

Steps Performed at Containment Level 2:

Precipitate the RNA at −70° C. for at least 30 minutes—can do this stepovernight.

Spin the precipitated RNA down at 13,000 rpm for 10 minutes. Remove thesupernatant and wash the pellet in 70% ethanol. Repeat centrifugation.

Remove the 70% ethanol and air-dry the pellet. Dissolve the pellet inRNAse free water.

Freeze the RNA at −70° C. to store it.

EXAMPLE 3 Isolation of Genomic DNA from Mycobacterium tuberculosis Grownin Chemostat Culture. DNA Then Used to Generate Cy3 or Cy5 Labelled DNAfor Use as a Control in Microarray Experiments

Materials and Methods

-   Beads 0.5 mm in diameter-   Bead beater-   Bench top centrifuge-   Platform rocker-   Heat block-   Falcon 50 ml centrifuge tubes-   Sorvall RC-5C centrifuge-   250 ml polypropylene centrifuge pots.-   Screw capped eppendorf tubes-   Pipettes 1 ml, 200 μl, 10 ml, 5 ml    Breaking Buffer-   50 mM Tris HCl pH 8.0-   110 mM EDTA-   100 mM NaCl    Procedure    Mechanical Disruption of M. tuberculosis Cells    -   150 ml of chemostat cells (O.D of 2.5 at 540 nm) are spun down        at 15,000 rpm for 15 minutes in 250 ml polypropylene pots using        centrifuge Sorvall RC-5C.    -   The supernatant is discarded.    -   Cells are re-suspended in 5 ml of breaking buffer in a 50 ml        Falcon tube and centrifuged at 15,000 rpm for a further 15        minutes.    -   The supernatant is removed and additional breaking buffer is        added at a volume of 5 ml. Beads are used to disrupt the cells.        These are used at a quantity of 1 ml of beads for 1 ml of cells.        Place the sample into the appropriate sized chamber. Place in        the bead beater and secure the outer unit (containing ice) and        process at the desired speed for 30 seconds.    -   Allow the beads to settle for 10 minutes and transfer cell        lysate to a 50 ml Falcon centrifuge tube.    -   Wash beads with 2-5 ml of breaking buffer by pipetting washing        buffer up and down over the beads.    -   Add this washing solution to the lysate in the falcon tube        Removal of Proteins and Cellular Components    -   Add 0.1 volumes of 10% SDS and 0.01 volumes proteinase K.    -   Mix by inversion and heat at 55° C. in a heat block for 2-3        hours    -   The resulting mix should be homogenous and viscous. Additional        SDS may be added to assist here to bring the concentration up to        0.2%    -   Add an equal volume of phenol/chloroform/Isoamyl alcohol in the        ratio: 25/24/1.    -   Gently mix on a platform rocker until homogenous    -   Spin down at 3,000 rpm for 20 minutes    -   Remove the aqueous phase and place in a fresh tuber    -   Extract the aqueous phase with an equal volume of chloroform to        remove traces of cell debris and phenol. Chloroform extractions        may need to be repeated to remove all the debris.    -   Precipitate, the DNA with 0.3 M sodium acetate and an equal        volume of isopropanol.    -   Spool as much DNA as you can with a glass rod    -   Wash the spooled DNA in 70% ethanol followed by 100% ethanol    -   Leave to air dry    -   Dissolve the DNA in sterile deionized water (500 μl)    -   Allow DNA to dissolve at 4° C. for approximately 16 hours.    -   Add RNase 1 (500 U) to the dissolved DNA    -   Incubate for 1 hour at 37° C.    -   Re-extract with an equal volume of phenol/chloroform followed by        a chloroform extraction and precipitate as before    -   Spin down the DNA at 13,000 rpm    -   Remove the supernatant and wash the pellet in 70% ethanol    -   Air dry    -   Dissolve in 200-500 μl of sterile water.

EXAMPLE 4 Preparation of Cy3 or Cy5 Labelled DNA from DNA

a) Prepare one Cy3 or one Cy5 Labelled DNA Sample per Microarray Slide.

Each sample:

DNA 2-5 μg Random primers (3 μg/μl) 1 μl H₂O to 41.5 μl

Heat at 95° C. for 5 min, snap cool on ice and briefly centrifuge.

-   Add to each:-   10× REact 2 buffer . . . 5 μl-   dNTPs (5 mM dA/G/TTP, 2 mM dCTP) . . . 1 μl-   Cy3 OR Cy5 dCTP . . . 1.5 μl-   Klenow (5 U/μl) . . . 1 μl-   Incubate at 37° C. in dark for 90 min.    b) Prehybridize Slide

Mix the prehybridization solution in a Coplin jar and incubate at 65° C.during the labelling reaction to equilibrate.

-   -   Prehybridization: 20×SSC . . . 8.75 ml (3.5×SSC)    -    20% SDS . . . 250 μl (0.1% SDS)    -    BSA (100 mg/ml) . . . 5 ml (10 mg/ml)    -    H₂O . . . to 50 ml

Incubate the microarray slide in the pre-heated prehybridizationsolution at 65° C. for 20 min. Rinse slide thoroughly in 400 ml H₂O for1 min followed by rinse in 400 ml propan-2-ol for 1 min and centrifugeslide in 50 ml centrifuge tube at 1,500 rpm for 5 min to dry. Storeslide in dark, dust-free box until hybridization (<1 h).

c) Purify Cy3/Cy5 Labelled DNA—Qiagen MinElute Purification

-   -   Combine Cy3 and Cy5 labelled DNA samples in single tube and add        500 μl Buffer PB.    -   Apply to MinElute column in collection tube and centrifuge at        13,000 rpm for 1 min.    -   Discard flow-through and place MinElute column back into same        collection tube.    -   Add 500 μl Buffer PE to MinElute column and centrifuge at 13,000        rpm for 1 min.    -   Discard flow-through and place MinElute column back into same        collection tube.    -   Add 250 μl Buffer PE to MinElute column and centrifuge at 13,000        rpm for 1 min.    -   Discard flow-through and place MinElute column back into same        collection tube.    -   Centrifuge at 13,000 rpm for an additional 1 min to remove        residual ethanol.    -   Place the MinElute column into a fresh 1.5 ml tube.    -   Add 10.5 μl H₂O to the centre of the membrane and allow to stand        for 1 min.    -   Centrifuge at 13,000 rpm for 1 min.

EXAMPLE 5 Preparation of Cy3 or Cy5 Label cDNA from RNA

a) Prepare One Cy3 and One Cy5 Labelled cDNA Sample Per Microarray Slide

Each sample:

-   RNA . . . 2-10 μg-   Random primers (3 μg/μl). . . 1 μl-   H₂O . . . to 11 μl

Heat at 95° C. for 5 min, snap cool on ice and briefly centrifuge.

-   Add to each: 5ÎFirst Strand Buffer . . . 5 μl-   DTT (100 mM) . . . 2.5 μl-   dNTPs (5 mM dA/GTTP, 2 mM dCTP) . . . 2.3 μl-   Cy3 OR Cy5 dCTP . . . 1.7 μl-   SuperScript II (200 U/μl) . . . 2.5 μl

Incubate at 25° C. in dark for 10 min followed by 42° C. in dark for 90min.

-   b) Prehybridize slide

Mix the prehybridization solution in a Coplin jar and incubate at 65° C.during the labelling reaction to equilibrate.

-   -   Prehybridization: 20×SSC . . . 8.75 ml (3.5×SSC)    -    20% SDS . . . 250 μl (0.1% SDS)    -    BSA (100 mg/ml) . . . 5 ml (10 mg/ml)    -    H₂O . . . to 50 ml

Incubate the microarray slide in the pre-heated prehybridizationsolution at 65° C. for 20 min. Rinse slide thoroughly in 400 ml H₂O for1 min followed by rinse in 400 ml propan-2-ol for 1 min and centrifugeslide in 50 ml centrifuge tube at 1,500 rpm for 5 min to dry. Storeslide in dark, dust-free box until hybridization (<1 h).

c) Purify Cy3/Cy5 Labelled cDNA—Qiagen MinElute Purification

-   -   Combine Cy3 and Cy5 labelled DNA samples in single tube and add        250 μl Buffer PB.    -   Apply to MinElute column in collection tube and centrifuge at        13,000 rpm for 1 min.    -   Discard flow-through and place MinElute column back into same        collection tube.    -   Add 500 μl Buffer PE to MinElute column and centrifuge at 13,000        rpm for 1 min.    -   Discard flow-through and place MinElute column back into same        collection tube.    -   Add 250 μl Buffer PE to MinElute column and centrifuge at 13,000        rpm for 1 min.    -   Discard flow-through and place MinElute column back into same        collection tube.    -   Centrifuge at 13,000 rpm for an additional 1 min to remove        residual ethanol.    -   Place the MinElute column into a fresh 1.5 ml tube.    -   Add 10.5 μl H₂O to the centre of the membrane and allow to stand        for 1 min.    -   Centrifuge at 13,000 rpm for 1 min.

EXAMPLE 6 Hybridize Slide with Cy3/Cy5 Labelled cDNA

Place the prehybridize microarray slide in the hybridization cassetteand add two 15 ml aliquots of H₂O to the wells in the cassette. Mixresuspended Cy3/Cy5 labelled cDNA sample with hybridization solution.

-   -   Hybridization: Cy3/Cy5 labelled cDNA sample . . . 10.5 ml    -    20×SSC . . . 3.2 ml (4×SSC)    -    2% SDS . . . 2.3 ml (0.3% SDS)

Heat hybridization solution at 95° C. for 2 min. Do not snap cool on icebut allow to cool slightly and briefly centrifuge. Pipette thehybridization solution onto the slide at the edge of the arrayed areaavoiding bubble formation. Using forceps carefully drag the edge of acover slip along the surface of the slide towards the arrayed area andinto the hybridization solution at the edge of the array. Carefullylower the cover slip down over the array avoiding any additionalmovement once in place. Seal the hybridization cassette and submerge ina water bath at 60° C. for 16-20 h.

Wash slide.

Remove microarray slide from hybridization cassette and initially washslide carefully in staining trough of Wash A to remove cover slip. Oncecover slip is displaced place slide(s) in slide rack and continueagitating in Wash A for a further 2 min.

-   -   Wash A: 20×SSC . . . 20 ml. (1×SSC)    -    20% SDS . . . 1 ml (0.05% SDS)    -    H₂O . . . to 400 ml

Transfer slide(s) to a clean slide rack and agitate in first trough ofWash B for 2 min. Wash in second trough of Wash B with agitation for 2min.

-   -   Wash B (×2):20×SSC . . . 1.2 ml (0.06×SSC).)    -    H₂O . . . to 400 ml

Place slide into a 50 ml centrifuge tube and centrifuge at 1500 rpm for5 mins to dry slide, and then scan fluorescence using a ScanArray 3000dual-laser confocal scanner and analyze data.

EXAMPLE 7 Preparation of the Arrays

PCR-amplified products are generated from M. tuberculosis genomic DNAusing ORF-specific primers. Each gene of the genome is represented.These are spotted in a grid onto a standard glass microscope slide usinga BioRobotics microgrid robot (MWG Biotech) at a resolution of >4000spots/cm².

EXAMPLE 8 Scanning and Analysis of Data

The slides were scanned using an Affymetrix 428 scanner.

Dual fluorescence is used, allowing simultaneous detection of two cDNAsamples. The output of the arrays is read using a confocal laser scanner(Affimetrix 428 scanner from MWG Biotech). More detailed information canbe found in Mujumdar, R. B. (1993) Bioconjugate Chemistry, 4(2), pp.105-111; Yu, H. (1994) Nucl. Acids Res. 22, pp. 3226-3232; and Zhu, Z.(1994) Nucl. Acids Res. 22, pp. 3418-3422.

The raw data were initially analyzed in software known as ImaGene, whichwas supplied with the scanner. The scanned images were then transferredto another software package known as GeneSpring. This is a very powerfultool, which draws information from many databases allowing the completeanalysis of the expression of each gene.

EXAMPLE 9 Delete One or More of the Genes from M. tuberculosis in Orderto Attenuate its Virulence While Retaining Immunogenicity

One or more genes that are identified may be disrupted using allelicexchange. In brief, the gene of interest is cloned with 1-2 kb offlanking DNA either side and is inactivated by deletion of part of thecoding region and insertion of an antibiotic resistance marker, such ashygromycin.

The manipulated fragment is then transferred to a suitable suicidevector e.g. pPR23 and is transformed into the wild-type parent strain ofM. tuberculosis. Mutants are recovered by selecting for antibioticresistant strains. Genotypic analysis (Southern Blotting with a fragmentspecific to the gene of interest) is performed on the selected strainsto confirm that the gene has been disrupted.

The mutant strain is then studied to determine the effect of the genedisruption on the phenotype. In order to use it as a vaccine candidateit would be necessary to demonstrated attenuated virulence. This can bedone using either a guinea pig or mouse model of infection. Animals areinfected with the mutant strain and the progression of disease ismonitored by determining the bacterial load in different organs, inparticular the lung and spleen, at specific time points post infection,typically up to 16 weeks.

Comparison is made to animals infected with the wild-type strain whichshould have a significantly higher bacterial load in the differentorgans. Long-term survival studies and histopathology can also be usedto assess virulence and pathogenicity.

Once attenuated virulence has been established, protection andimmunogenicity studies can be performed to assess the potential of thestrain as a vaccine. Suitable references for allelic exchange andpreparation of TB mutants are McKinney et al., 2000 and Pelicic et al.,1997, [1, 2].

EXAMPLE 10 Select One or More of the Genes Identifiable by the PresentInvention, Which Encode Proteins that are Immunogenic, and Put Them intoBCG or an Attenuated Strain of M. tuberculosis to Enhance its OverallImmunogenicity

The gene of interest is amplified from the M. tuberculosis genome byPCR. The amplified product is purified and cloned into a plasmid(pMV306) that integrates site specifically into the mycobacterial genomeat the attachment site (attB) for mycobacteriophage L5 [3].

BCG is transformed with the plasmid by electroporation, which involvesdamaging the cell envelope with high voltage electrical pulses,resulting in uptake of the DNA. The plasmid integrates into the BCGchromosome at the attB site generating stable recombinants. Recombinantsare selected and are checked by PCR or Southern blotting to ensure thatthe gene has been integrated. The recombinant strain is then used forprotection studies.

EXAMPLE 11 Use of Recombinant Carriers Such as Attenuated Salmonella andthe Vaccinia Virus to Express and Present TB Genes

One of the best examples of this type of approach is the use of ModifiedVaccinia virus Ankara (MVA) [4]. The gene of interest is cloned into avaccinia virus shuttle vector, e.g. pSC11. Baby Hamster Kidney (BHK)cells are then infected with wild-type MVA and are transfected with therecombinant shuttle vector. Recombinant virus is then selected using asuitable selection marker and viral plaques, selected and purified.

Recombinant virus is normally delivered as part of a prime-boost regimewhere animals are vaccinated initially with a DNA vaccine encoding theTB genes of interest under the control of a constitutive promoter. Theimmune response is boosted by administering recombinant MVA carrying thegenes of interest to the animals at least 2 weeks later.

EXAMPLE 12 Sub-unit Vaccines Containing a Single Peptide/Protein or aCombination of Proteins

To prepare sub-unit vaccines with one or more peptides or proteins it isfirst of all necessary to obtain a supply of protein or peptide toprepare the vaccine. Up to now, this has mainly been achieved inmycobacterial studies by purifying proteins of interest from TB culture.However, it is becoming more common to clone the gene of interest andproduce a recombinant protein.

The coding sequence for the gene of interest is amplified by PCR withrestriction sites inserted at the N terminus and C terminus to permitcloning in-frame into a protein expression vector such as pET-15b. Thegene is inserted behind an inducible promoter such as lacZ. The vectoris then transformed into E. coli which is grown in culture. Therecombinant protein is over-expressed and is purified.

One of the common purification methods is to produce a recombinantprotein with an N-terminal His-tag. The protein can then be purified onthe basis of the affinity of the His-tag for metal ions on a Ni-NTAcolumn after which the His-tag is cleaved. The purified protein is thenadministered to animals in a suitable, adjuvant [5].

EXAMPLE 13 Plasmid DNA Vaccines Carrying One or More of the IdentifiedGenes

DNA encoding a specific gene is amplified by PCR, purified and insertedinto specialized vectors developed for vaccine development, such aspVAX1. These vectors contain promoter sequences, which direct strongexpression of the introduced DNA (encoding candidate antigens) ineukaryotic cells (e.g. CMV or SV40 promoters), and polyadenlyationsignals (e.g. SV40 or bovine growth hormone) to stabilize the mRNAtranscript.

The vector is transformed into E. coli and transformants are selectedusing a marker, such as kanamycin resistance, encoded by the plasmid.The plasmid is then recovered from transformed colonies and is sequencedto check that the gene of interest is present and encoded properlywithout PCR generated mutations.

Large quantities of the plasmid is then produced in E. coli and theplasmid is recovered and purified using commercially available kits(e.g. Qiagen Endofree-plasmid preparation). The vaccine is thenadministered to animals for example by intramuscular injection in thepresence or absence of an adjuvant.

EXAMPLE 14 Preparation of DNA Expression Vectors

DNA vaccines consist of a nucleic acid sequence of the present inventioncloned into a bacterial plasmid. The plasmid vector pVAX1 is commonlyused in the preparation of DNA vaccines. The vector is designed tofacilitate high copy number replication in E. coli and high leveltransient expression of the peptide of interest in most mammalian cells.For details see manufacturers protocol for pVAX1 (catalog no. V260-20).

The vector contains the following elements:—

-   -   Human cytomegalovirus immediate-early (CMV) promoter for        high-level expression in a variety of mammalian cells    -   T7 promoter/priming site to allow in vitro transcription in the        sense orientation and sequencing through the insert    -   Bovine growth hormone (BGH) polyadenylation signal for efficient        transcription termination and polyadenylation of mRNA    -   Kanamycin resistance gene for selection in E. coli    -   A multiple cloning site    -   pUC origin for high-copy number replication and growth in E.        coli    -   BGH reverse priming site to permit sequencing through the insert

Vectors may be prepared by means of standard recombinant techniqueswhich are known in the art, for example Sambrook et al., (1989). Keystages in preparing the vaccine are as follows:

-   -   The gene of interest is ligated into pVAX1 via one of the        multiple cloning sites    -   The ligation mixture is then transformed into a competent E.        coli strain (e.g. TOP10) and LB plates containing 50 μg/ml        kanamycin are used to select transformants.    -   Clones are selected and may be sequenced to confirm the presence        and orientation of the gene of interest.    -   Once the presence of the gene has been verified, the vector can        be used to transfect a mammalian cell line to check for protein        expression. Methods for transfection are known in the art and        include, for example, electroporation, calcium phosphate, and        lipofection.    -   Once peptide expression has been confirmed, large quantities of        the vector can be produced and purified from the appropriate        cell host, e.g. E. coli.

pVAX1 does not integrate into the host chromosome. All non-essentialsequences have been removed to minimize the possibility of integration.When constructing a specific vector, a leader sequence may be includedto direct secretion of the encoded protein when expressed inside theeukaryotic cell.

Other examples of vectors that have been used are V1Jns.tPA and pCMV4(Lefevre et al., 2000 and Vordermeier et al., 2000).

Expression vectors may be used that integrate into the genome of thehost, however, it is more common and more preferable to use a vectorthat does not integrate. The example provided, pVAX1, does notintegrate. Integration would lead to the generation of a geneticallymodified host which raises other issues.

EXAMPLE 15 RNA Vaccine

As discussed on page 15 of U.S. Pat. No. 5,783,386, one approach is tointroduce RNA directly into the host.

Thus, the vector construct (Example 10) may be used to generate RNA invitro and the purified RNA then injected into the host. The RNA wouldthen serve as a template for translation in the host cell. Integrationwould not occur.

Another option is to use an infectious agent such as the retroviralgenome carrying RNA corresponding to the gene of interest. Here you willget integration into the host genome

Another option is the use of RNA replicon vaccines which can be derivedfrom virus vectors such as Sindbis virus or Semliki Forest virus. Thesevaccines are self-replicating and self-limiting and may be administeredas either RNA or DNA which is then transcribed into RNA replicons invivo. The vector eventually causes lysis of the transfected cellsthereby reducing concerns about integration into the, host genome.Protocols for RNA vaccine construction are detailed in Cheng et al.,(2001).

EXAMPLE 16 Diagnostic Assays Based on Assessing T Cell Responses

For a diagnostic assay based on assessing T cell responses it would besufficient to obtain a sample of blood from the patient. Mononuclearcells (monocytes, T and B lymphocytes) can be separated from the bloodusing density gradients such as Ficoll gradients.

Both monocytes and B-limphocytes are able to present antigen, althoughless efficiently than professional antigen presenting cells (APCs) suchas dendritic cells. The latter are more localized in lymphoid tissue.

The simplest approach would be to add antigen to the separatedmononuclear cells and incubate for a week and then assess the amount ofproliferation. If the individual had been exposed to the antigenpreviously through infection, then T-cell clones specific to the antigenshould be more prevalent in the sample and should respond.

It is also possible to separate the different cellular populationsshould it be desired to control the ratio of T cells to APC's.

Another variation of this type of assay is to measure cytokineproduction by the responding lymphocytes as a measure of response. TheELISPOT assay described below in Example 17 is a suitable example ofthis variation.

EXAMPLE 17 Detection of Latent Mycobacteria

A major problem for the control of tuberculosis is the presence of alarge reservoir of asymptomatic individuals infected with tuberclebacilli. Dormant bacilli are more resistant to front-line drugs.

The presence of latent mycobacteria-associated antigen may be detectedindirectly either by detecting antigen specific antibody or T-cells inblood samples.

The following method is based on the method described in Lalvani et al.(2001) in which a secreted antigen, ESAT-6, was identified as beingexpressed by members of the M. tuberculosis complex but is absent fromM. bovis BCG vaccine strains and most environmental mycobacteria. 60-80%of patients also have a strong cellular immune response to ESAT-6. Anex-vivo ELISPOT assay was used to detect ESAT-6 specific T cells.

As applied to the present invention:

A 96 well plate is coated with cytokine (e.g. interferon-γ,IL-2)-specific antibody. Peripheral blood monocytes are then isolatedfrom patient whole blood and are applied to the wells.

Antigen (ie. one of the peptides, fragments, derivatives or variants ofthe present invention) is added to stimulate specific T cells that maybe present and the plates are incubated for 24 h. The antigen stimulatescytokine production which then binds to the specific antibody.

The plates are washed leaving a footprint where antigen-specific T cellswere present.

A second antibody coupled with a suitable detection system, e.g. enzyme,is then added and the number of spots are enumerated after theappropriate substrate has been added.

The number of spots, each corresponding to a single antigen-specific Tcell, is related to the total number of cells originally added.

The above Example also describes use of an antigen that may be used todistinguish TB infected individuals from BCG vaccinated individuals.This could be used in a more discriminative diagnostic assay.

EXAMPLE 18 In vitro Model for Mycobacterial Persistence Under the JointConditions of Carbon-starvation and Oxygen-limitation ( Variation onExamples 1-7)

Materials and Methods

Strain

Studies were performed with M. tuberculosis strain H37Rv (NCTC cat. No.7416)—a representative strain of M. tuberculosis. Stock cultures weregrown on Middlebrook 7H10+OADC for 3 weeks at 37±2° C.

Culture Medium

Persistence cultures were established in Middlebrook 7H9 mediumsupplemented with Middlebrook ADC enrichment, 0.2% Tween 80 and 0.2%glycerol (see below). The medium was prepared with high quality waterfrom a Millipore water purification system and filter sterilized bypassage through a 0.1 um pore size cellulose acetate membrane filtercapsule (Sartorius Ltd). The pH was adjusted to 6.6 with concentratedhydrochloric acid.

Middlebrook 7H10+OADC agar was used to prepare inoculum cultures,enumerate the number of culturable bacteria in samples, and to assessculture purity.

Culture System

The culture system described in WO00/52139, operated as a batchfermenter, was employed for this Example.

Culture experiments were performed in a one liter glass vessel operatedat a working volume of 750 ml. The culture was agitated by a magneticbar placed in the culture vessel coupled to a magnetic stirrerpositioned beneath the vessel. Culture conditions were continuouslymonitored by an Anglicon Microlab Fermentation System (Brighton Systems,Newhaven), linked to sensor probes inserted into the culture throughsealed ports in the top plate. The oxygen concentration was monitoredwith a galvanic oxygen electrode (Uniprobe, Cardiff) and was controlledthrough sparging the culture with a mixture of air and oxygenfree-nitrogen. Temperature was monitored by an Anglicon temperatureprobe, and maintained by a heating pad positioned beneath the culturevessel. Culture pH was measured using an Ingold pH electrode(Mettler-Toledo, Leicester).

Inoculation and Culture

The vessel was filled with 750 ml of sterile culture medium andparameters were allowed to stabilize at 37° C.±2 C, pH 6.9±0.3 and adissolved oxygen tension of approximately 70% air saturation. A denseinoculum suspension was prepared by resuspending Middlebrook agarcultures, grown at 37° C. for 3 week, in sterile deionized water. Theinoculum was aseptically transferred to the culture vessel, to providean initial culture turbidity of approximately 0.25 at 540 nm. Theculture was maintained at 37° C. with an agitation rate of 500 to 750rpm.

After inoculation, the dissolved oxygen tension (DOT) of the culture wasmaintained at approximately 40% air saturation at 37° C. until theculture had entered early exponential growth. The DOT was then loweredin increments down to 1% air saturation over a six day period (FIG. 3).The culture was then maintained at a DOT of 0-5% until 50 days afterinoculation and samples were removed regularly to monitor growth andsurvival, nutrient utilization and gene expression.

Growth and Survival

Bacterial growth and survival was assessed by determining the number ofviable cells in the culture system at specific time points. This wasachieved by preparing a decimal dilution series of the sample in sterilewater and plating 100 μl aliquots onto Middlebrook 7H10+OADC plates. Theplates were incubated at 37° C. for up to 4 weeks before enumerating thenumber of colonies formed.

Nutrient Utilization

Glycerol is the primary carbon and energy source present in Middlebrook7H9 medium with ADC, 0.2% Tween and Glycerol. The rate at which glycerolwas utilized was determined using the Glycerol Determination Kit Cat.No. 148 270 Boehringer Mannheim.

Microarray Experiments

RNA was extracted from culture samples collected at different timepoints during the experiment. A fluorescently-labelled cDNA was thentranscribed from each sample of RNA. The cDNA was labelled by theincorporation of either Cy3 or Cy5 labelled dCTP (Dyes are supplied byAmersham Pharmacia Biotech).

Whole M. tuberculosis genome arrays were prepared from M. tuberculosisgenomic DNA using ORF-specific primers. PCR products corresponding toeach ORF were spotted in a grid onto a standard glass microscope slideusing a BioRobotics microgrid robot (MWG Biotech) at a resolutionof >4000 spots/cm².

Arrays were supplied by Dr P Butcher, St George's Hospital MedicalSchool London.

In each microarray experiment a whole genome array was hybridized withlabelled cDNA from one culture sample (Test sample). Each array was alsohybridized with control DNA incorporating a different Cy dye andprepared from DNA extracted from M. tuberculosis strain H37Rv (controlsample). Each array was scanned, using an Affymetrix 428 scanner, at twodifferent wavelengths corresponding to the excitation maxima of each dyeand the intensity of the emitted light was recorded. The raw data wasprocessed by ImaGene software before performing comparative analysisusing GeneSpring.

Results

Analysis of viable count data indicated that the culture grewexponentially until 10 to 12 days post infection (FIG. 4). As theculture entered stationary phase, viability started to decline andcontinued to decline steadily over the duration of the study. After 40days in stationary phase, approximately 0.1% of the culture was stillculturable on Middlebrook agar. The rate of glycerol utilization wasslower than observed in the culture established under aerobicconditions, indicating that the metabolic activity of the low-oxygenculture was restricted by limited oxygen availability. Nevertheless, theprincipal carbon and energy source was depleted within 15 days afterinoculation (FIG. 2).

Samples were collected for microarray analysis as outlined. The geneexpression profiles for samples collected at day 5 and 50 were compared.Three arrays were prepared for each sample and the test data wasnormalized against the control data on each chip. The normalized datafor each set of arrays was then averaged and the two data sets werecompared. Those genes that were expressed at least 1.5-fold, preferablyat least 5-fold higher at day 50 relative to day 5 were selected. Thegene list was then added to those genes identified under “nutrient-starving” conditions, and the complete set listed in Table 2.

Liquid medium formulation for persistence cultures—Middlebrook 7H9medium supplemented with ADC, 0.2% Tween 80 and 0.2% Glycerol

Composition Per Liter:

Composition per litre: Na₂HPO₄ 2.5 g KH₂PO₄ 1.0 g Monosodium glutamate0.5 g (NH4)₂SO₄ 0.5 g Sodium citrate 0.1 g MgSO₄.7H₂O 0.05 g Ferricammonium citrate 0.04 g CuSO₄.5H₂O 1.0 mg Pyridoxine 1.0 mg ZnSO₄.7H₂O1.0 mg Biotin 0.5 mg CaCl₂.2H₂O 0.5 mg Middlebrook ADC enrichment 100 mlGlycerol 2.0 ml Tween 80 2.0 ml Middlebrook ADC enrichment - per 100 mlBovine serum albumin 5.0 g Glucose 2.0 g Catalase 3.0 mgMicroarray Protocols1. RNA Extraction from M. tuberculosis for Microarray AnalysisMaterials and Methods

-   -   Trizol (Life Technologies) formulation of phenol and guanidine        thiocyanate.    -   GTC lysis solution containing: 5 M guanidine thiocyanate, 0.5%        N-lauryl sarcosine, 25 mM tri-sodium citrate, 0.1 M        2-mercaptoeathanol, and 0.5% Tween 80.    -   Chloroform    -   Isopropanol    -   3 M sodium acetate    -   70% Ethanol    -   microfuge    -   ribolyser    -   Sterile plasticware-Falcon tubes, screw capped eppendorfs,        gilson tips—all RNase free    -   Glassware—baked at 160° C. for at least 16 hours        Method    -   Steps performed at Containment level 3; within a Class III        microbiological safety cabinet.    -   Remove 10 or 20 ml of culture (10⁹/ml) and immediately add this        to 4 volumes of GTC lysis buffer in a plastic specimen pot. Seal        the pot tightly.    -   Incubate the cells in GTC lysis buffer for 1 hour at room        temperature. Surface decontaminate the plastic pot with 5%        Hycolin for 5 minutes. Transfer the sample to the pass box and        place it into a plastic carry tin with a sealable lid. Close the        container securely and transport it to a non-toxic cabinet CL3        cabinet.    -   Equally distribute the lysis mixture between Falcon tubes. Place        these tubes into centrifuge buckets and seal the buckets        tightly. Surface-decontaminate the buckets for 5 minutes with 5%        Hycolin. Then transfer them to the centrifuge (Baird and Tatlock        Mark IV refrigerated bench-top centrifuge). Spin the tubes at        3,000 rpm for 30 minutes.    -   Return the unopened buckets to the cabinet. Remove the        centrifuge tubes and pour the supernatant into a waste bottle        for GTC lysis buffer.    -   Resuspend each pellet in 5 ml of Trizol (formulation of phenol        and GTC cat No. 15596-026). The manufacturers guidelines        recommend lysing cells by repetitive pipetting. Although this        action alone will not lyse M. tuberculosis, it is important to        completely resuspend the pellet in Trizol.    -   Transfer 1 ml of cells into each FastRNA tube and ribolyse them        at power setting 6.5 for 45 seconds.    -   Leave the tubes to incubate at room temperature for 5 minutes.    -   Remove the aqueous layer from each tube and add this to 200 μl        of chloroform in a screw-capped eppendorf tube. Shake each tube        vigorously for about 15 seconds. Incubate for 2-3 minutes at        room temperature.    -   Spin the tubes at 13,000 rpm for 15 minutes. Following        centrifugation, the liquid separates into red phenol/chloroform        phase, an interface, and a clear aqueous phase.    -   Carefully remove the aqueous phase and transfer it to fresh        eppendorf tubes containing 500 μl of chloroform/isoamyl alcohol        (24:1). Spin the tubes at 13,000 rpm for 15 minutes.    -   Transfer the aqueous phase to eppendorf tubes containing 50 μl        of sodium acetate and 500 μl of isopropanol.    -   Surface decontaminate the eppendorf tubes with 5% Hycolin for 5        minutes.    -   Steps performed at Containment level 2:    -   Precipitate the RNA at −70° C. for at least 30 minutes        (optionally overnight).    -   Spin the precipitated RNA down at 13,000 rpm for 10 minutes.        Remove the supernatant and wash the pellet in 70% ethanol.        Repeat centrifugation.    -   Remove the 70% ethanol and air-dry the pellet. Dissolve the        pellet in RNAse free water.    -   Freeze the RNA at −70° C. to store it.        2. Isolation of Genomic DNA from Mycobacterium tuberculosis        Grown in Chemostat Culture. DNA then Used to Generate Cy3 or Cy5        Labelled DNA for Use as a Control in Microarray Experiments        Materials and Methods

-   Beads 0.5 mm in diameter

-   Bead beater

-   Bench top centrifuge

-   Platform rocker

-   Heat block

-   Falcon 50 ml centrifuge tubes

-   Sorvall RC-5 C centrifuge

-   250 ml polypropylene centrifuge pots.

-   Screw capped eppendorf tubes

-   Pipettes 1 ml, 200 μl, 10 ml, 5 ml    Breaking Buffer

-   50 mM Tris HCL pH 8.0

-   10 M EDTA

-   100 mM NaCl    Procedure    Mechanical Disruption of Mtb Cells    -   150 ml of chemostat cells (O.D of 2.5 at 540 nm) are spun down        at 15,000 rpm for 15 minutes in 250 ml polypropylene pots using        centrifuge Sorvall RC-5C.    -   The supernatant is discarded    -   Cells are re-suspended in 5 ml of breaking buffer in a 50 ml        Falcon tube and centrifuged at 15,000 rpm for a further 15        minutes.    -   The supernatant is removed and additional breaking buffer is        added at a volume of 5 ml. Beads are used to disrupt the cells.        These are used at a quantity of 1 ml of beads for 1 ml of cells.        Place the sample into the appropriate sized chamber. Place in        the bead beater and secure the outer unit (containing ice) and        process at the desired speed for 30 seconds.    -   Allow the beads to settle for 10 minutes and transfer cell        lysate to a 50 ml Falcon centrifuge tube    -   Wash beads with 2-5 ml of breaking buffer by pipetting washing        buffer up and down over the beads.    -   Add this washing solution to the lysate in the falcon tube.

Removal of proteins and cellular components.

-   -   Add 0.1 volumes of 10% SDS and 0.01 volumes proteinase K.    -   Mix by inversion and heat at 55° C. in a heat block for 2-3        hours.    -   The resulting mix should be homogenous and viscous. If it isn't        then add more SDS to bring the concentration up to 0.2%    -   Add an equal volume of phenol/chloroform/Isoamyl alcohol in the        ratio: 25/24/1.    -   Gently mix on a platform rocker until homogenous    -   Spin down at 3,000 rpm for 20 minutes    -   Remove the aqueous phase and place in a fresh tube    -   Extract the aqueous phase with an equal volume of chloroform to        remove traces of cell debris and phenol. Chloroform extractions        may need to be repeated to remove all the debris.    -   Precipitate the DNA with 0.3 M sodium acetate and an equal        volume of isopropanol.    -   Spool as much DNA as you can with a glass rod    -   Wash the spooled DNA in 70% ethanol followed by 100% ethanol    -   Leave to air dry    -   Dissolve the DNA in sterile deionized water (500 μl)    -   Allow DNA to dissolve at 4° C. for approximately 16 hours.    -   Add RNase 1 (50 U.) to the dissolved DNA    -   Incubate for 1 hour at 37° C.    -   Re-extract with an equal volume of phenol/chloroform followed by        a chloroform extraction and precipitate as before    -   Spin down the DNA at 13,000 rpm    -   Remove the supernatant and wash the pellet in 70% ethanol    -   Air dry    -   Dissolve in 200-500 μl of sterile water.        3. Preparation of Cy3 or Cy5 Labelled DNA from DNA        a) Prepare One Cy3 or One Cy5 Labelled DNA Sample Per Microarray        Slide.

-   Each sample DNA 2-5 μg

-   Random primers (3 μg/μl) 1 μl

-   H₂O to 41.5 μl

-   Heat at 95° C. for 5 min, snap cool on ice and briefly centrifuge.

-   Add to each: 10*REact 2 buffer . . . 5 μl

-   dNTPs (5 mM dA/G/TTP, 2 mM dCTP) . . . 1 μl

-   Cy3 OR Cy5 dCTP . . . 1.5 μl

-   Klenow (5 U/μl) . . . 1 μl

-   Incubate at 37° C. in dark for 90 min.    b) Prehybridize Slide

Mix the prehybridization solution in a Coplin jar and incubate at 65° C.during the labelling reaction to equilibrate.

Prehybridization: 20×SSC 8.75 ml (3.5×SSC)

-   20% SDS 250 μl (0.1% SDS)-   BSA (100 mg/ml) 5 ml (10 mg/ml)-   H₂O to 50 ml

Incubate the microarray slide in the pre-heated prehybridizationsolution at 65° C. for 20 min. Rinse slide thoroughly in 400 ml H₂O for1 min followed by rinse in 400 ml propan-2-ol for 1 min and centrifugeslide in 50 ml centrifuge tube at 1,500 rpm for 5 min to dry. Storeslide in dark, dust-free box until hybridization (<1 h).

c) Purify Cy3/Cy5 Labelled DNA—Qiagen MinElute Purification

-   -   Combine Cy3 and Cy5 labelled DNA samples in single tube and add        500 μl Buffer PB.    -   Apply to MinElute column in collection tube and centrifuge at        13,000 rpm for 1 min.    -   Discard flow-through and place MinElute column back into same        collection tube.    -   Add 500 μl Buffer PE to MinElute column and centrifuge at 13,000        rpm for 1 min.    -   Discard flow-through and place MinElute column back into same        collection tube.    -   Add 250 μl Buffer PE to MinElute column and centrifuge at 13,000        rpm for 1 min.    -   Discard flow-through and place MinElute column back into same        collection tube.    -   Centrifuge at 13,000 rpm for an additional 1 min to remove        residual ethanol.    -   Place the MinElute column into a fresh 1.5 ml tube.    -   Add 10.5 μl H₂O to the centre of the membrane and allow to stand        for 1 min.    -   Centrifuge at 13,000 rpm for 1 min.        4. Preparation of Cy3 or Cy5 Label cDNA from RNA        a) Prepare One Cy3 and One Cy5 Labelled cDNA Sample Per        Microarray Slide.

-   Each sample: RNA . . . 2-10 μg

-   Random primers (3 μg/μl) . . . . 1 μl

-   H₂O . . . to 11 μl

-   Heat at 95° C. for 5 min, snap cool on ice and briefly centrifuge.

-   Add to each: 5*First Strand Buffer . . . 5 μl

-   DTT (100 mM) . . . 2.5 μl

-   dNTPs (5 mM dA/G/TTP, 2 mM dCTP) . . . 2.3 μl

-   Cy3 OR Cy5 dCTP . . . 1.7 μl

-   SuperScript II (200 U/μl) . . . 2.5 μl

Incubate at 25° C. in dark for 10 min followed by 42° C. in dark for 90min.

b) Prehybridize Slide

Mix the prehybridization solution in a Coplin jar and incubate at 65° C.during the labelling reaction to equilibrate.

Prehybridization: 20×SSC 8.75 ml (3.5×SSC)

-   20% SDS 250 μl (0.1% SDS)-   BSA (100 mg/ml) 5 ml (10 mg/ml)-   H₂O to 50 ml

Incubate the microarray slide in the pre-heated prehybridizationsolution at 65° C. for 20 min. Rinse slide thoroughly in 400 ml H₂O for1 min followed by rinse in 400 ml propan-2-ol for 1min and centrifugeslide in 50 ml centrifuge tube at 1500 rpm for 5 min to dry. Store slidein dark, dust-free box until hybridization (<1 h).

c) Purify Cy3/Cy5 Labelled cDNA—Qiagen MinElute Purification

-   -   Combine Cy3 and Cy5 labelled DNA samples in single tube and add        250 μl Buffer PB.    -   Apply to MinElute column in collection tube and centrifuge at        13,000 rpm for 1 min.    -   Discard flow-through and place MinElute column back into same        collection tube.    -   Add 500 μl Buffer PE to MinElute column and centrifuge at 13,000        rpm for 1 min.    -   Discard flow-through and place MinElute column back into same        collection tube.    -   Add 250 μl Buffer PE to MinElute column and centrifuge at 13,000        rpm for 1 min.    -   Discard flow-through and place MinElute column back into same        collection tube.    -   Centrifuge at 13,000 rpm for an additional 1 min to remove        residual ethanol.    -   Place the MinElute column into a fresh 1.5 ml tube.    -   Add 10.5 μl H₂O to the centre of the membrane and allow to stand        for 1 min.    -   Centrifuge at 13,000 rpm for 1 min.        5. Hybridize slide with Cy3/Cy5 labelled cDNA/DNA

Place the prehybridized microarray slide in the hybridization cassetteand add two 15 μl d aliquots of H₂O to the wells in the cassette. Mixresuspended Cy3/Cy5 labelled cDNA sample with hybridization solution.

Hybridization:

-   Cy3/Cy5 labelled cDNA sample 10.5 μl-   20×SSC 3.2 μl (4×SSC)-   2% SDS 2.3 μl (0.3% SDS)

Heat hybridization solution at 95° C. for 2 min. Do NOT snap cool on icebut allow to cool slightly and briefly centrifuge. Pipette thehybridization solution onto the slide at the edge of the arrayed areaavoiding bubble formation. Using forceps carefully drag the edge of acover slip along the surface of the slide towards the arrayed area andinto the hybridization solution at the edge of the array. Carefullylower the cover slip down over the array avoiding any additionalmovement once in place. Seal the hybridization cassette and submerge ina water bath at 65° C. for 16-20 h.

Wash Slide

Remove microarray slide from hybridization cassette and initially washslide carefully in staining trough of Wash A, preheated to 65° C., toremove cover slip. Once cover slip is displaced place slide(s) in sliderack and continue agitating in Wash A for a further 2 min.

-   -   Wash A: 20×SSC . . . 20 ml (1×SSC)    -    20% SDS . . . 1 ml (0.05% SDS)    -    H₂O . . . to 400 ml

Transfer slide(s) to a clean slide rack and agitate in first trough ofWash B for 2 min. Wash in second trough of Wash B with agitation for 2min.

-   -   Wash B (×2):20×SSC . . . 1.2 ml (0.06×SSC)    -    H₂O . . . to 400 ml

Place slide into a 50 ml centrifuge tube and centrifuge at 1500 rpm for5 mins to dry slide and then scan fluorescence.

TABLE 2 Genes induced or up-regulated under nutrient-starvingconditions, or under nutrient-starving and oxygen-limiting conditions.Gene Assigned function SEQ. ID. NO. Rv0021c 2-nitropropane dioxygenase1, 2 Rv0029 3, 4 Rv0076c peptide with a membrane-spanning domain at itsC-terminus 5, 6 Rv0111 acetyltransferase 7, 8 Rv0161 oxidoreductase  9,10 Rv0212c transcriptional regulator 11, 12 Rv0228 acyl transferase 13,14 Rv0260c two-component response regulator 15, 16 Rv0311 17, 18 Rv0322UDP-glucose dehyrogenase 19, 20 Rv0325 21, 22 Rv0389phosphoribosylglycinamide formyltransferase 23, 24 Rv0390 25, 26 Rv039527, 28 Rv0480c 29, 30 Rv0493c 31, 32 Rv0534c 1,4-dihydroxy-2-naphthoateoctaprenyl 33, 34 Rv0557 35, 36 Rv0614 37, 38 Rv0621 peptide containinga membrane-spanning region 39, 40 Rv0622 peptide containing amembrane-spanning region 41, 42 Rv0697 gmc-type oxidoreductase 43, 44Rv0698 45, 46 Rv0736 47, 48 Rv0751c 3-hydroxyisobutyrate dehydrogenase;methylmalonate semialdehyde 49, 50 dehydrogenase Rv0775 51, 52 Rv0776c53, 54 Rv0785 dehydrogenase 55, 56 Rv0790c 57, 58 Rv0794c mercuricreductase; glutathione reductase; dihydrolipoamide dehydrogenase 59, 60Rv0795 transposase 61, 62 Rv0836c 63, 64 Rv0837c 65, 66 Rv0840c prolineiminopeptidase; prolyl aminopeptidase 67, 68 Rv0849 integral membranetransport protein; quinolone efflux pump 69, 70 Rv0917 glycine betainetransporter 71, 72 Rv978c 73, 74 Rv1051c 75, 76 Rv1056 77, 78 Rv1089 79,80 Rv1146 membrane protein 81, 82 Rv1147 phosphatidylethanolamineN-methyltransferase 83, 84 Rv1370c transposase 85, 86 Rv1371 membraneprotein 87, 88 Rv1372 chalcone synthase 2 89, 90 Rv1373 sulfotransferase91, 92 Rv1429 93, 94 Rv1455 95, 96 Rv1482c 97, 98 Rv1496  99, 100Rv1526c glycosyl transferase 101, 102 Rv1528c PKS-associated protein103, 104 Rv1552 fumarate reductase flavoprotein 105, 106 Rv15698-amino-7-oxononanoate synthase; aminotransferase class-II pyridoxal-107, 108 phosphate Rv1573 phage phiRv1 protein 109, 110 Rv1577cbacteriophage HK97 prohead protease; phage phiRv1 protein 111, 112Rv1670 113, 114 Rv1725c 115, 116 Rv1730 penicillin-binding protein 117,118 Rv1763 transposase 119, 120 Rv1765c 121, 122 Rv1777 cytochrome p450123, 124 Rv1806 125, 126 Rv1866 fatty acyl-CoA racemase 127, 128 Rv1917c129, 130 Rv1939 nitrilotriacetate monooxygenase 131, 132 Rv2013transposase 133, 134 Rv2027c histidine kinase response regulator 135,136 Rv2086 transposase 137, 138 Rv2087 transposase 139, 140 Rv2089cpepQ; peptidase 141, 142 Rv2091c peptide containing a transmembraneregion 143, 144 Rv2093c TatC component of twin-arginine translocationprotein export system 145, 146 Rv2105 transposase 147, 148 Rv2168ctransposase 149, 150 Rv2242 151, 152 Rv2282c LysR transcriptionregulator 153, 154 Rv2292c 155, 156 Rv2310 excisionase 157, 158 Rv2322cornithine aminotransferase 159, 160 Rv2323c 161, 162 Rv2332 malateoxidoreductase 163, 164 Rv2400c thiosulphate-binding protein 165, 166Rv2414c 167, 168 Rv2437 169, 170 Rv2478c 171, 172 Rv2486 enoyl-coAhydratase 173, 174 Rv2505c acyl-CoA synthetase 175, 176 Rv2529methyltransferase 177, 178 Rv2596 179, 180 Rv2847c multifunctionalenzyme; siroheme synthase 181, 182 Rv3635 transmembrane protein 183, 184Rv2643 membrane protein 185, 186 Rv2648 transposase 187, 188 Rv2655c189, 190 Rv2684 transmembrane protein; arsenical pump 191, 192 Rv2687cregulatory protein 193, 194 Rv2690c transport protein; permease 195, 196Rv2800 glutaryl 7-aca acylase 197, 198 Rv2812 transposase 199, 200Rv2813 secretion pathway protein 201, 202 Rv2835csn-glycerol-3-phosphate transport system permease protein 203, 204Rv2874 integral membrane protein 205, 206 Rv2877c mercury resistanceprotein 207, 208 Rv2943 transposase 209, 210 Rv2998 211, 212 Rv3015c213, 214 Rv3022c 215, 216 Rv3039c enoyl-CoA hydratase/isomerase 217, 218Rv3016c acyl-CoA dehydrogenase 219, 220 Rv3064c 221, 222 Rv3097cesterase; lipase 223, 224 Rv3107c dehydrogenase 225, 226 Rv3162c 227,228 Rv3178 229, 230 Rv3184 transposase 231, 232 Rv3315c cytidinedeaminase 233, 234 Rv3322c methyltransferase 235, 236 Rv3351c 237, 238Rv3352c oxidoreductase 239, 240 Rv3373 enoyl-CoA hydratase (crotonase)241, 242 Rv3439c 243, 244 Rv3446c 245, 246 Rv3447c membrane protein 247,248 Rv3450c 249, 250 Rv3467 251, 252 Rv3505 acyl-CoA dehydrogenase 253,254 Rv3540c lipid-transfer protein 255, 256 Rv3546 acetyl-CoAC-acetyltransferase 257, 258 Rv3550 enoyl-CoA hydratase/isomerase 259,260 Rv3552 261, 262 Rv3565 aminotransferase 263, 264 Rv3596c hydrolase265, 266 Rv3606c 2-amino-4-hydroxy-6-hydroxymethyldihydropterinepyrophosphokinase 267, 268 Rv3637 transposase 269, 270 Rv3660c 271, 272Rv3745c 273, 274 Rv3903c 275, 276 Rv0039c 277, 278 Rv0903c 279, 280Rv2745c 281, 282

REFERENCES

-   1. McKinney, J. D. et al., Persistence of Mycobacterium tuberculosis    in macrophages and mice requires the glyoxylate shunt enzyme    isocitrate lyase [see comments]. Nature, 2000. 406(6797): p. 735-8.-   2. Pelicic, V., et al., Efficient allelic exchange and transposon    mutagenesis in Mycobacterium tuberculosis. Proc Natl Acad Sci    USA, 1997. 94(20): p. 10955-60.-   3. Lee, M. H., et al., Site-specific integration of    mycobacteriophage L5: integration proficient vectors for    Mycobacterium smegmatis, Mycobacterium tuberculosis, and bacille    Calmette-Guerin. Proc Natl Acad Sci USA, 1991. 88(8): p. 3111-5.-   4. McShane, H., et al., Enhanced immunogenicity of CD4(+) t-cell    responses and protective efficacy of a DNA-modified vaccinia virus    Ankara prime-boost vaccination regimen for murine tuberculosis.    Infect Immun, 2001. 69(2): p. 681-6.-   5. Movahedzadeh, F., M. J. Colston, and E. O. Davis,    Characterization of Mycobacterium tuberculosis LexA: recognition of    a Cheo (Bacillus-type SOS) box. Microbiology, 1997. 143(Pt 3): p.    929-36.

ADDITIONAL REFERENCES

-   Cunningham, A. F. and. C. L. Spreadbury. 1998. Mycobacterial    stationary phase induced by low oxygen tension: cell wall thickening    and localization of the 16-kilodalton alpha-crystallin homolog. J.    Bacteriol. 180:801-808.-   Lalvani, A. et al., 2001. Enhanced contact tracing and spatial    tracking of Mycobacterium tuberculosis infection by enumeration of    antigen-specific T cells. The Lancet 357:2017-2021.-   Rook, G. A. W. and B. R. Bloom. 1994. Mechanisms of pathogenesis in    tuberculosis, pp 460-485. In B. R. Bloom (ed),    Tuberculosis—pathogenesis, on and control. ASM Press, Washington    D.C.-   Wayne, L. G. 1994. Dormancy of Mycobacterium tuberculosis and    latency of disease. Eur. J. Clin. Microbiol. Infect. Dis.    13:908-914.-   Wayne, L. G. and L. G. Hayes. 1996. An in vitro model for    sequential, study of shift-down of Mycobacterium tuberculosis    through two stages of non-replicating persistence. Infect. Immun.    64:2062-2069.-   Wayne, L. G. and K. Lin. 1982. Glyoxylate metabolism and adaptation    of Mycobacterium tuberculosis to survival under anaerobic    conditions. Infect. Immun. 37:1042-1049.-   Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular    cloning: a laboratory manual, 2nd ed. Cold Spring Harbour Laboratory    Press, Cold Spring Harbour, N.Y.-   Lefever, P., O. Denis, L. De Wit, A. Tanghe, P. Vandenbussche, J.    Content, and K. Huygen. 2000. Cloning of the gene encoding a    22-kilodalton cell surface antigen of Mycobacterium bovis BCG and    analysis of its potential for DNA vaccination against tuberculosis.    Infection and Immunity. 68:1040-1047.-   Vordermeire, H. M., P. J. Cockle, A. O. Whelan, S. Rhodes, M. A.    Chambers, D. Clifford, K. Huygen, R. Tascon, D. Lowrie, M. J.    Colston, and R. G. Hewinson. 2000. Effective DNA vaccination of    cattle with the mycobacterial antigens MPB83 and MPB70 does not    compromise the specificity of the comparative intradermal tuberculin    skin test. Vaccine. 19:1246-1255.-   Cheng, W., C. Hung, C. Chai, K. Hsu, L. He, C. Rice, M. Ling, and T.    Wu. 2001. Enhancement of Sindbis virus self-replicating RNA vaccine    potency by linkage of Mycobacterium tuberculosis heat shock protein    70 gene to an antigen. J. Immunol. 166:6218-6226.

1. An isolated M. tuberculosis peptide selected from the groupconsisting of SEQ ID NOs: 7, 55, 125 and 129 or a variant thereof havingat least 70% amino acid homology therewith, or a fusion protein thereof,wherein said fusion protein or variant has a common antigeniccross-reactivity to said isolated peptide; wherein the peptide isencoded by an M. tuberculosis gene the expression of which is induced orup regulated under culture conditions that are nutrient starving andwhich maintain mycobacterial latency, said conditions being obtainableby batch fermentation of an M. tuberculosis micobacterium for at least40 days post inoculation, when compared with culture conditions that arenot nutrient starving and which support exponential growth of saidmycobacterium.
 2. A therapeutic agent for combating mycobacterialinfections, comprising an M. tuberculosis peptide selected from thegroup consisting of SEQ ID NOs: 7, 55, 125 and 129, or a fusion proteinthereof, wherein said fusion protein has a common antigeniccross-reactivity to said peptide; wherein the peptide is encoded by anM. tuberculosis gene the expression of which is induced or up regulatedunder culture conditions that are nutrient starving and which maintainmycobacterial latency, said conditions being obtainable by batchfermentation of an M. tuberculosis micobacterium for at least 40 dayspost inoculation, when compared with culture conditions that are notnutrient starving and which support exponential growth of saidmycobacterium.
 3. A method of treating an M. tuberculosis infection, byadministering to a patient an M. tuberculosis peptide selected from thegroup consisting of SEQ ID NOs: 7, 55, 125 and 129, or a fusion proteinthereof, wherein said fusion protein has a common antigeniccross-reactivity to said peptide; wherein the peptide is encoded by anM. tuberculosis gene the expression of which is induced or up regulatedunder culture conditions that are nutrient starving and which maintainmycobacterial latency, said conditions being obtainable by batchfermentation of an M. tuberculosis micobacterium for at least 40 dayspost inoculation, when compared with culture conditions that are notnutrient starving and which support exponential growth of saidmycobacterium.
 4. A method of diagnosing a mycobacterial infection,comprising the steps: (i) incubating a biological sample with anisolated M. tuberculosis peptide selected from the group consisting ofSEQ ID NOs: 7, 55, 125 and 129 wherein the peptide is encoded by an M.tuberculosis gene the expression of which is induced or up regulatedunder culture conditions that are nutrient starving and which maintainmycobacterial latency, said conditions being obtainable by batchfermentation of an M. tuberculosis micobacterium for at least 40 dayspost inoculation, when compared with culture conditions that are notnutrient starving and which support exponential growth of saidmycobacterium; and (ii) detecting antibodies to mycobacteria, whereinsaid antibodies bind to said isolated peptide, and thereby detectingmycobacterial infection.
 5. A diagnostic reagent for identifying amycobacterial infection, comprising an isolated M. tuberculosis peptideselected from the group consisting of SEQ ID NOs: 7, 55, 125 and 129, ora fusion protein thereof, wherein said fusion protein has a commonantigenic cross-reactivity to said peptide; wherein the peptide isencoded by an M. tuberculosis gene the expression of which is induced orup regulated under culture conditions that are nutrient starving andwhich maintain mycobacterial latency, said conditions being obtainableby batch fermentation of an M. tuberculosis micobacterium for at least40 days post inoculation, when compared with culture conditions that arenot nutrient starving and which support exponential growth of saidmycobacterium.
 6. An isolated peptide, wherein said peptide is: (i) afragment of an M. tuberculosis peptide selected from the groupconsisting of SEQ ID NOs: 7, 55, 125 and 129, wherein said fragment hasat least 35 amino acids; (ii) a variant of (i) having at least 70% aminoacid homology therewith; or (iii) a fusion protein of (i) or (ii);wherein said fragment, variant or fusion protein has a common antigeniccross-reactivity to said M. tuberculosis peptide; wherein the M.tuberculosis peptide is encoded by an M. tuberculosis gene theexpression of which is induced or up regulated under culture conditionsthat are nutrient starving and which maintain mycobacterial latency,said conditions being obtainable by batch fermentation of an M.tuberculosis micobacterium for at least 40 days post inoculation, whencompared with culture conditions that are not nutrient starving andwhich support exponential growth of said mycobacterium.
 7. A therapeuticagent for combating mycobacterial infections, comprising an isolatedpeptide, wherein said isolated peptide is: (i) a variant having at least90% amino acid homology to a peptide selected from the group consistingof: (a) an M. tuberculosis peptide selected from the group consisting ofSEQ ID NOs: 7, 55, 125 and 129; and (b) a fragment of (a) having atleast 35 amino acid residues; or (ii) a fusion protein of (i); whereinsaid fragment, variant or fusion protein has a common antigeniccross-reactivity to said M. tuberculosis peptide; and wherein the M.tuberculosis peptide is encoded by an M. tuberculosis gene theexpression of which is induced or up regulated under culture conditionsthat are nutrient starving and which maintain mycobacterial latency,said conditions being obtainable by batch fermentation of an M.tuberculosis micobacterium for at least 40 days post inoculation, whencompared with culture conditions that are not nutrient starving andwhich support exponential growth of said mycobacterium.
 8. A diagnosticreagent for identifying a mycobacterial infection, comprising anisolated peptide, wherein said isolated peptide is: (i) a variant havingat least 90% amino acid homology to a peptide selected from the groupconsisting of: (a) an M. tuberculosis peptide selected from the groupconsisting of SEQ ID NOs: 7, 55, 125 and 129; and (b) a fragment of (a)having at least 35 amino acid residues; or (ii) a fusion protein of (i);wherein said variant, fragment or fusion protein has a common antigeniccross-reactivity to said M. tuberculosis peptide; and wherein the M.tuberculosis peptide is encoded by an M. tuberculosis gene theexpression of which is induced or up regulated under culture conditionsthat are nutrient starving and which maintain mycobacterial latency,said conditions being obtainable by batch fermentation of an M.tuberculosis micobacterium for at least 40 days post inoculation, whencompared with culture conditions that are not nutrient starving andwhich support exponential growth of said mycobacterium.
 9. A therapeuticagent for combating mycobacterial infections, comprising an isolatedpeptide, wherein said peptide is: (i) a fragment of an M. tuberculosispeptide selected from the group consisting of SEQ ID NOs: 7, 55, 125 and129, wherein said fragment has at least 35 amino acid residues; or (ii)a fusion protein of (i); wherein said fragment, or fusion protein has acommon antigenic cross-reactivity to said M. tuberculosis peptide;wherein the M. tuberculosis peptide is encoded by an M. tuberculosisgene the expression of which is induced or up regulated under cultureconditions that are nutrient starving and which maintain mycobacteriallatency, said conditions being obtainable by batch fermentation of an M.tuberculosis micobacterium for at least 40 days post inoculation, whencompared with culture conditions that are not nutrient starving andwhich support exponential growth of said mycobacterium.
 10. A method oftreating an M. tuberculosis infection, by administering to a patient anisolated peptide, wherein said peptide is: (i) a fragment of an M.tuberculosis peptide selected from the group consisting of SEQ ID NOs:7, 55, 125 and 129, wherein said fragment has at least 35 amino acids;or (ii) a fusion protein of (i); wherein said fragment or fusion proteinhas a common antigenic cross-reactivity to said M. tuberculosis peptide;wherein the M. tuberculosis peptide is encoded by an M. tuberculosisgene the expression of which is induced or up regulated under cultureconditions that are nutrient starving and which maintain mycobacteriallatency, said conditions being obtainable by batch fermentation of an M.tuberculosis micobacterium for at least 40 days post inoculation, whencompared with culture conditions that are not nutrient starving andwhich support exponential growth of said mycobacterium.
 11. A method ofdiagnosing a mycobacterial infection, comprising the steps of: (i)incubating a biological sample with an isolated peptide, wherein saidisolated peptide is a fragment of an M. tuberculosis peptide selectedfrom the group consisting of SEQ ID NOs: 7, 55, 125 and 129, whereinsaid fragment has at least 35 amino acids; wherein said fragment, has acommon antigenic cross-reactivity to said M. tuberculosis peptide;wherein the M. tuberculosis peptide is encoded by an M. tuberculosisgene the expression of which is induced or up regulated under cultureconditions that are nutrient starving and which maintain mycobacteriallatency, said conditions being obtainable by batch fermentation of an M.tuberculosis micobacterium for at least 40 days post inoculation, whencompared with culture conditions that are not nutrient starving andwhich support exponential growth of said mycobacterium; and (ii)detecting antibodies to mycobacteria, wherein said antibodies bind tosaid fragment, and thereby detecting mycobacterial infection.
 12. Adiagnostic reagent for identifying a mycobacterial infection, comprisingan isolated peptide, wherein said peptide is: (i) a fragment of an M.tuberculosis peptide selected from the group consisting of SEQ ID NOs:7, 55, 125 and 129, wherein said fragment has at least 35 amino acids;or (ii) a fusion protein of (i); wherein said fragment or fusion proteinhas a common antigenic cross-reactivity to said M. tuberculosis peptide;wherein the M. tuberculosis peptide is encoded by an M. tuberculosisgene the expression of which is induced or up regulated under cultureconditions that are nutrient starving and which maintain mycobacteriallatency, said conditions being obtainable by batch fermentation of an M.tuberculosis micobacterium for at least 40 days post inoculation, whencompared with culture conditions that are not nutrient starving andwhich support exponential growth of said mycobacterium.
 13. A medicamentfor reducing the severity or intensity of a mycobacterial infection, orfor preventing mycobacterial disease progression, by administering to apatient an M. tuberculosis peptide selected from the group consisting ofSEQ ID NOs: 7, 55, 125 or 129, or a fusion protein thereof, wherein saidfusion protein has a common antigenic cross-reactivity to said peptide;wherein the peptide is encoded by an M. tuberculosis gene the expressionof which is induced or up regulated under culture conditions that arenutrient starving and which maintain mycobacterial latency, saidconditions being obtainable by batch fermentation of an M. tuberculosismicobacterium for at least 40 days post inoculation, when compared withculture conditions that are not nutrient starving and which supportexponential growth of said mycobacterium.
 14. A method of reducing theseverity or intensity of a mycobacterial infection, or of preventingmycobacterial disease progression, by administering to a patient anisolated peptide, wherein said isolated peptide is: (i) a fragment of anM. tuberculosis peptide selected from the group consisting of SEQ IDNOs: 7, 55, 125 and 129, wherein said fragment has at least 35 aminoacids; or (ii) a fusion protein of (i); wherein said fragment or fusionprotein has a common antigenic cross-reactivity to said M. tuberculosispeptide; and wherein the M. tuberculosis peptide is encoded by an M.tuberculosis gene the expression of which is induced or up regulatedunder culture conditions that are nutrient starving and which maintainmycobacterial latency, said conditions being obtainable by batchfermentation of an M. tuberculosis micobacterium for at least 40 dayspost inoculation, when compared with culture conditions that are notnutrient starving and which support exponential growth of saidmycobacterium.
 15. An isolated peptide, wherein said peptide is: (i) afragment of a polypeptide, wherein said polypeptide is a variant havingat least 90% amino acid homology with an isolated M. tuberculosispeptide selected from the group consisting of SEQ ID NOs: 7, 55, 125 and129, wherein said fragment has at least 35 amino acids residues; or (ii)a fusion protein of (i); wherein said fragment, variant or fusionprotein has a common antigenic cross-reactivity to said M. tuberculosispeptide; and wherein the M. tuberculosis peptide is encoded by an M.tuberculosis gene the expression of which is induced or up regulatedunder culture conditions that are nutrient starving and which maintainmycobacterial latency, said conditions being obtainable by batchfermentation of an M. tuberculosis micobacterium for at least 40 dayspost inoculation, when compared with culture conditions that are notnutrient starving and which support exponential growth of saidmycobacterium.
 16. A therapeutic agent for combating mycobacterialinfections, comprising an isolated peptide, wherein said isolatedpeptide is: (i) a fragment of a polypeptide, wherein said polypeptide isa variant having at least 90% amino acid homology with an isolated M.tuberculosis peptide selected from the group consisting of SEQ ID NOs:7, 55, 125 and 129, wherein said fragment has at least 35 amino acids;or (ii) a fusion protein of (i); wherein said fragment, variant orfusion protein has a common antigenic cross-reactivity to said isolatedM. tuberculosis peptide; and wherein the M. tuberculosis peptide isencoded by an M. tuberculosis gene the expression of which is induced orup regulated under culture conditions that are nutrient starving andwhich maintain mycobacterial latency, said conditions being obtainableby batch fermentation of an M. tuberculosis micobacterium for at least40 days post inoculation, when compared with culture conditions that arenot nutrient starving and which support exponential growth of saidmycobacterium.
 17. A diagnostic reagent for identifying a mycobacterialinfection, comprising an isolated peptide, wherein said peptide is: (i)a fragment of a polypeptide, wherein said polypeptide is a varianthaving at least 90% amino acid homology with an isolated M. tuberculosispeptide selected from the group consisting of SEQ ID NOs: 7, 55, 125 and129, wherein said fragment has at least 35 amino acids; or (ii) a fusionprotein of (i); wherein said fragment, variant or fusion protein has acommon antigenic cross-reactivity to said isolated M. tuberculosispeptide; and wherein the M. tuberculosis peptide is encoded by an M.tuberculosis gene the expression of which is induced or up regulatedunder culture conditions that are nutrient starving and which maintainmycobacterial latency, said conditions being obtainable by batchfermentation of an M. tuberculosis micobacterium for at least 40 dayspost inoculation, when compared with culture conditions that are notnutrient starving and which support exponential growth of saidmycobacterium.